- [Resource Hub](/) - [Ebook](/?resource-type=ebook#library) - Tissue Dissociation Guide Protocol Resource # Comprehensive Tissue Dissociation Protocol Guide 57+ tissue types for single-cell and single-nuclei workflows. Community-validated protocols with Singulator-optimized methods from Precision Cell Systems. [Explore the Singulator](https://precisioncellsystems.com/products-singulator/) [Request a Quote](https://precisioncellsystems.com/request-a-quote/) Protocol Navigator 57+ Tissues 43 Cell Isolation Protocols 42 Nuclei Isolation Protocols 100+ Published References 66 Singulator Protocols [Tissue to Cells Guidelines](#cells-section) [Tissue to Nuclei Guidelines](#nuclei-section) Important: Tissue Variability Notice As with all tissue dissociation workflows, outcomes depend on tissue quality, species, age, region, fixation or storage history, and donor variability. The protocols, expected yields, and benchmarks described in this guide represent commonly observed results under recommended conditions and should be treated as starting points. Actual performance may vary. Always validate with your specific tissue source before committing precious samples. Cell Isolation ## Tissue-to-Cells Protocols 43 tissue types with step-by-step enzymatic and mechanical dissociation protocols for single-cell analysis. Select Tissue Type Adipose Adrenal Gland Aorta / Vasculature Bladder Bone / Cartilage Bone Marrow Brain Breast (Normal and Tumor) C. elegans Cochlea / Inner Ear Colon / Intestine Cornea Dental Pulp Dorsal Root Ganglia / Peripheral Nerve Drosophila Endometrium / Uterus Esophagus Heart iPSC-Derived Tissues Kidney Liver Lung (Normal and Tumor) Lymph Node Mammary Gland Muscle (Skeletal) Organoids and Spheroids Ovary Pancreas Placenta Prostate Retina / Eye Salivary Gland Skin / Dermis Spinal Cord Spleen Stomach Tendon / Ligament Testis Thymus Thyroid Trachea / Airway Tumor (General Solid Tumors) Zebrafish ### Adipose #### Overview Adipose tissue is composed primarily of adipocytes (fat cells), which are large (50-200 um), lipid-filled, and buoyant. The stromal vascular fraction (SVF) contains preadipocytes, endothelial cells, smooth muscle cells, pericytes, fibroblasts, mesenchymal stem cells, macrophages, T cells, and other immune cells. Collagenase digestion followed by separation of the floating adipocyte layer from the SVF pellet is the most commonly used approach. The lipid content creates unique debris challenges. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase I 1-2 mg/mL Sigma C0130 Standard for adipose Collagenase II 1 mg/mL Worthington LS004176 Alternative DNase I 50 U/mL Roche Anti-clumping HBSS or PBS 1x Various Wash buffer BSA 1-2% Various Blocks nonspecific enzyme binding ACK Lysis Buffer 1x Various RBC removal from SVF #### Step-by-Step Protocol - Tissue preparation (10 min): Mince adipose tissue into 1-3 mm pieces with scissors. Wash 3x with warm PBS to remove blood. - Enzymatic digestion (30-60 min): Incubate in Collagenase I (1 mg/mL) + DNase I (50 U/mL) in HBSS + 2% BSA at 37 degrees C with shaking (120-150 rpm). Tissue becomes cloudy as adipocytes release lipid. - Separation (5 min): Centrifuge at 300g for 5 min. Three layers form: floating adipocytes (top), liquid (middle), SVF pellet (bottom). Aspirate adipocyte layer and liquid. - RBC lysis (5 min): Resuspend SVF in ACK buffer for 3-5 min. Quench with HBSS. - Filtration (5 min): Filter through 100 um then 70 um strainers to remove undigested fragments. - Counting (5 min): Count SVF cells with AO/PI. #### Expected Results - SVF yield : 200,000-1,000,000 cells per gram of adipose - Viability : 80-95% for SVF cells - Common cell types recovered : Preadipocytes, endothelial cells, pericytes, MSCs, fibroblasts, macrophages (M1 and M2), T cells, B cells, NK cells, mast cells - Processing time : 60-90 minutes #### Tips and Tricks - Warm digestion is typically required : Cold protocols are generally ineffective for adipose tissue, as lipid content and extracellular matrix composition limit enzymatic access at low temperatures. - Collagenase lot testing is important : Adipose dissociation quality can vary significantly between collagenase lots. Test each lot before committing precious samples. - Lipid debris removal : After SVF isolation, debris can be further removed by passing through a 40 um strainer or brief Percoll gradient. - Multi-sample considerations : For high-throughput adipose processing requiring 8+ simultaneous samples, batch-processing systems may be preferred. The Singulator excels when standardized, walk-away automation and reproducibility are prioritized over throughput. #### Troubleshooting Problem Possible Cause Solution Lipid contamination of SVF Incomplete adipocyte removal Additional wash after centrifugation; use 40 um strainer Low SVF yield Insufficient digestion Extend time to 60 min; increase collagenase to 2 mg/mL Clumping DNA from dead cells Add DNase I; use wider bore pipette tips Poor viability Excessive mechanical force Reduce agitation speed; gentle trituration only #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : User-supplied collagenase cocktail via Single-Shot mechanism Notes : Adipose tissue processing is supported on the Singulator but does not have a pre-formulated reagent. The platform's open reagent design allows loading of custom collagenase cocktails. Note that the Singulator processes 1 sample at a time (S100) or up to 2 samples (S200), which may limit throughput for high-volume adipose studies. Source : Precision Cell Systems Application Data #### References [1] protocols.io: Miltenyi Biotec: Adipose Tissue Dissociation. U Minnesota. https://www.protocols.io/view/miltenyi-biotec-adipose-tissue-dissociation-univer-ctpywmpw ### Adrenal Gland #### Overview The adrenal gland has two distinct regions: the outer cortex (zona glomerulosa, fasciculata, reticularis producing aldosterone, cortisol, and androgens) and the inner medulla (chromaffin cells producing catecholamines). The cortex requires collagenase digestion; chromaffin cells are more fragile. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1 mg/mL Worthington LS004188 Standard for adrenal DNase I 50 U/mL Roche Anti-clumping #### Step-by-Step Protocol - Tissue preparation (5 min): Remove fat. For separate cortex/medulla analysis, dissect under microscope. Mince into 1 mm pieces. - Enzymatic digestion (30-45 min): Collagenase IV (1 mg/mL) + DNase I (50 U/mL) at 37 degrees C with agitation. - Filtration and counting (10 min): Filter through 70 um strainer. Count with AO/PI. #### Expected Results - Cell yield : Variable; 100,000-500,000 cells per mouse adrenal - Viability : 80-90% - Common cell types recovered : Zona glomerulosa cells, zona fasciculata cells, zona reticularis cells, chromaffin cells, endothelial cells, immune cells #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform. #### References [1] Lyraki and Schedl (2021). The sexually dimorphic adrenal cortex: Implications for adrenal disease. International Journal of Molecular Sciences 22(9): 4889. ### Aorta / Vasculature #### Overview The aorta and large vessels have three layers: intima (endothelial cells), media (smooth muscle cells in an elastin-rich matrix), and adventitia (fibroblasts, vasa vasorum, immune cells). The elastic laminae and dense collagen make vascular tissue challenging to dissociate. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase II 1-2 mg/mL Worthington LS004176 For collagen matrix Elastase 0.5 mg/mL Worthington LS002279 Critical for elastic fibers DNase I 50 U/mL Roche Anti-clumping Soybean Trypsin Inhibitor 0.1 mg/mL Sigma T9128 Blocks released proteases #### Step-by-Step Protocol - Tissue preparation (10 min): Clean adventitial fat. Cut vessel open longitudinally. Mince into 1 mm pieces. - Enzymatic digestion (45-60 min): Collagenase II (1 mg/mL) + Elastase (0.5 mg/mL) + DNase I (50 U/mL) at 37 degrees C with agitation. - Filtration and counting (10 min): Filter through 70 um strainer. Count with AO/PI. #### Expected Results - Cell yield : Variable; depends on vessel size and length - Viability : 80-90% - Common cell types recovered : Endothelial cells, smooth muscle cells (dominant in media), adventitial fibroblasts, macrophages, T cells #### Singulator Protocol Singulator 100/200 -- Custom protocol with collagenase + elastase via open reagent platform. #### References [1] Kalluri et al. (2019). Single-Cell Analysis of the Normal Mouse Aorta Reveals Functionally Distinct Endothelial Cell Populations. Circulation 140(2): 147-163. ### Bladder #### Overview The urinary bladder has a transitional epithelium (urothelium) with umbrella cells, intermediate cells, and basal cells, underlaid by lamina propria and detrusor smooth muscle. The urothelium forms a tight barrier that resists enzymatic penetration. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1 mg/mL Worthington LS004188 For lamina propria Trypsin-EDTA 0.25% Gibco For urothelial dissociation DNase I 50 U/mL Roche Anti-clumping Dispase II 2.4 U/mL Roche Alternative for urothelial separation #### Step-by-Step Protocol - Tissue preparation (5 min): Eviscerate bladder. Cut open and spread flat. For separate layers, scrape urothelium or use Dispase II overnight to separate epithelium. - Enzymatic digestion (30-45 min): Collagenase IV (1 mg/mL) + DNase I (50 U/mL) at 37 degrees C for combined dissociation; or Trypsin-EDTA for urothelial cells. - Filtration and counting (10 min): Filter through 70 um strainer. Count with AO/PI. #### Expected Results - Cell yield : Variable depending on bladder size - Viability : 80-90% - Common cell types recovered : Urothelial cells (umbrella, intermediate, basal), fibroblasts, smooth muscle cells, endothelial cells, immune cells #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform. #### References [1] Yu Z, et al. (2019). Single-cell transcriptomic map of the human and mouse bladders. Journal of the American Society of Nephrology 30(11): 2159-2176. ### Bone / Cartilage #### Overview Bone and cartilage are mineralized/dense connective tissues. Bone dissociation for osteocytes requires decalcification or mechanical crushing, while cartilage (hyaline, elastic, fibrocartilage) is avascular and dense with proteoglycans and type II collagen. Both tissues yield relatively few cells and require extended enzymatic treatment. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase II 2-3 mg/mL Worthington LS004176 High concentration for dense ECM Pronase 1 mg/mL Roche 10165921001 For cartilage pre-treatment DNase I 100 U/mL Roche Critical EDTA 0.5 M, pH 7.4 Various For bone decalcification (hours to days) #### Step-by-Step Protocol (Cartilage) - Tissue preparation (10 min): Dice cartilage into 1 mm cubes. - Pronase pre-treatment (30 min): Incubate in Pronase (1 mg/mL) at 37 degrees C to strip proteoglycans. - Collagenase digestion (3-6 hours): Collagenase II (3 mg/mL) + DNase I (100 U/mL) at 37 degrees C with rotation. This is one of the longest enzymatic digestions required for any tissue. - Filtration and counting (10 min): Filter through 70 um strainer. Count with AO/PI. #### Expected Results - Cell yield : Low; 5,000-50,000 chondrocytes per gram of cartilage - Viability : 70-85% - Common cell types recovered : Chondrocytes (hypertrophic, resting, proliferating), osteoblasts (from bone), osteocytes (from decalcified bone), periosteal cells #### Singulator Protocol Bone and cartilage are among the most challenging tissues for any dissociation system. The extended digestion times (3-6 hours for cartilage) exceed typical Singulator protocol durations but could potentially be addressed through multiple sequential runs or extended custom protocols. #### References [1] Ji et al. (2019). Single-cell RNA-seq analysis reveals the progression of human osteoarthritis. Annals of the Rheumatic Diseases 78(1): 100-110. ### Bone Marrow #### Overview Bone marrow is a soft, spongy tissue found in the medullary cavity of bones. It is the primary site of hematopoiesis, containing hematopoietic stem cells (HSCs), progenitor cells, mature blood cells at various stages, mesenchymal stem cells (MSCs), osteoblasts, adipocytes, endothelial cells, and specialized macrophages. Unlike most solid tissues, bone marrow is already a semi-liquid tissue and requires minimal enzymatic digestion. The primary challenge is mechanical extraction from the bone, followed by RBC lysis and debris removal. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase I 0.5-1 mg/mL Sigma C0130 Only needed for stromal cell recovery DNase I 50 U/mL Roche Prevents clumping RPMI or IMDM 1x Various Base medium FBS 2-10% Various Cell protection ACK Lysis Buffer 1x Various CRITICAL -- bone marrow is predominantly RBCs Ficoll-Paque 1.077 g/mL GE Healthcare Mononuclear cell enrichment #### Step-by-Step Protocol - Bone marrow extraction (10-15 min): For mouse, cut femur/tibia at joints, flush marrow cavity with cold RPMI using 25G needle and syringe into a 15 mL tube. For human, aspirate with biopsy needle. - Mechanical dissociation (5 min): Triturate gently with 18G then 21G needle to break up clumps. Pass through 70 um cell strainer. - RBC lysis (5-10 min): Resuspend in 5-10 mL ACK buffer for 5 min at RT. May need 2 rounds given the high RBC content. Quench with 10 mL medium. - Optional Ficoll enrichment (30 min): Layer over Ficoll-Paque (1.077 g/mL). Centrifuge 400g for 30 min, brake off. Collect mononuclear cell interface. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : Typically ~10-50 million cells per mouse femur, depending on species strain, age, and flushing efficiency; human aspirates commonly yield substantially more - Viability : >95% for mononuclear cells - Common cell types recovered : HSCs, MPPs, CMPs, GMPs, MEPs, mature granulocytes, monocytes, erythroid progenitors, B cell progenitors, T cells, NK cells, MSCs (with enzymatic digestion), dendritic cells - Processing time : 30-45 minutes #### Tips and Tricks - Enzymatic digestion is optional : Standard hematopoietic cell preps need only mechanical flushing. Add collagenase only if stromal cells (MSCs, osteoblasts, endothelial cells) are needed. - Mouse vs. human : Mouse bone marrow is flushed from long bones; human is aspirated. Protocols differ significantly. - Speed matters for HSCs : HSC surface markers can change with prolonged processing. Minimize time from harvest to analysis. #### Troubleshooting Problem Possible Cause Solution Massive RBC contamination Single ACK lysis insufficient Repeat ACK lysis 2-3 times; or use Ficoll enrichment Low HSC yield Incomplete bone flushing Flush from both ends; crush bones in mortar for residual cells Clumping DNA from dead cells Add DNase I (50 U/mL) to flushing medium #### Singulator Protocol Bone marrow typically does not require the Singulator platform, as it is already a semi-liquid tissue. The Singulator is designed for solid tissue dissociation. However, for bone marrow biopsies (trephine biopsies with solid bone fragments), the Singulator could potentially process the tissue with a nuclei or cell isolation protocol using custom enzymes via the open reagent platform. #### References [1] Amend et al. (2016). Murine hind limb long bone dissection and bone marrow isolation. Journal of Visualized Experiments (110): e53936. ### Brain #### Overview Brain tissue presents unique dissociation challenges due to high lipid content (myelin), diverse cell populations (neurons, astrocytes, oligodendrocytes, microglia, endothelial cells), and the fragility of neuronal processes. The brain ECM is relatively soft compared to connective tissues but is enriched in hyaluronic acid, tenascins, and proteoglycans rather than fibrillar collagens. Papain is the most commonly used enzyme for brain dissociation because it typically cleaves broadly without the harsh effects of trypsin on neuronal surface markers. Adult brain tissue requires more aggressive dissociation than embryonic or neonatal brain due to increased myelination and ECM density. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Papain 20 U/mL Worthington LK003150 (Papain Dissociation System) Most commonly used enzyme for neural tissue; includes DNase I and ovomucoid inhibitor DNase I 100 U/mL Worthington (included in kit) Prevents clumping from released DNA Liberase TL 0.25-0.5 mg/mL Roche 05401020001 Alternative for gentle dissociation Collagenase IV 1 mg/mL Worthington LS004188 For tougher adult brain regions EBSS or HBSS 1x Various Balanced salt solution as base buffer Ovomucoid Inhibitor 10 mg/mL Worthington (in Papain kit) Stops papain activity Percoll/OptiPrep 22-40% gradient Various Myelin debris removal (critical for adult brain) #### Step-by-Step Protocol Manual Protocol (Papain Dissociation System) : - Tissue preparation (5 min): Dissect brain region of interest on ice in cold HBSS. Remove meninges if accessible. Mince tissue into 1-2 mm pieces with sterile scalpel or spring scissors. - Enzyme preparation (10 min): Reconstitute papain in EBSS with DNase I per Worthington kit instructions. Equilibrate to 37 degrees C. Activate papain with L-cysteine (1.1 mM final). - Enzymatic digestion (30-45 min): Incubate minced tissue in papain solution at 37 degrees C with gentle agitation (orbital shaker at 50-75 rpm or intermittent trituration every 10 min). - Trituration (5 min): Remove enzyme solution. Add ovomucoid inhibitor solution. Triturate 10-15 times with fire-polished Pasteur pipette (progressively narrower bore) until no visible clumps remain. - Filtration (3 min): Pass suspension through 70 um cell strainer, then 40 um strainer if desired. Wash strainer with cold HBSS. - Myelin removal (20-30 min): Layer cell suspension over 22% Percoll in PBS. Centrifuge at 500g for 15 min at 4 degrees C with brake off. Collect cell pellet, discard myelin layer at interface. - Wash and count (10 min): Wash pellet in cold HBSS + 0.04% BSA. Centrifuge 300g for 5 min. Resuspend in appropriate volume. Count with AO/PI or trypan blue. #### Expected Results - Cell yield : Typically ~10,000-200,000 cells per mg, depending on age, region, and species - Viability : 80-95% (trypan blue or AO/PI) - Common cell types recovered : Neurons, astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPCs), microglia, endothelial cells, pericytes - Processing time : 60-90 minutes total (manual); ~60 minutes on Singulator #### Tips and Tricks - Myelin removal is generally necessary for adult brain : Without Percoll or OptiPrep cleanup, myelin debris can clog microfluidic devices (10x Chromium, BD Rhapsody) and may significantly reduce capture efficiency. - Enzyme lot-to-lot variation : Papain activity varies between lots. Test each new lot on a small tissue sample before processing precious specimens. - RNase inhibitor for brain : Brain has lower RNase activity than pancreas or lung, but RNase inhibitor is still recommended for all brain dissociation workflows to protect RNA integrity. - Temperature matters for scRNA-seq : Cold (4 degrees C) protocols reduce dissociation-induced transcriptional changes but yield fewer cells. A 37 degrees C digest followed by rapid cooling is the most commonly used compromise. - Process brain nuclei samples first in multi-tissue experiments : Brain nuclei require the most downstream cleanup (myelin removal), so starting with brain allows time for those steps while processing other tissues. - For brain tumors : Use the Pan Tumor Reagent or Collagenase IV + DNase I rather than papain, as tumor ECM is more collagen-rich than normal brain. #### Troubleshooting Problem Possible Cause Solution Low viability Over-digestion with papain Reduce incubation time by 5-10 min; check papain activity Excessive debris Insufficient myelin removal Increase Percoll gradient concentration to 30%; ensure brake-off centrifugation Cell clumping Insufficient DNase I Increase DNase I to 200 U/mL; ensure fresh preparation Low neuronal yield Harsh trituration Use fire-polished pipettes; reduce trituration passes Poor scRNA-seq quality RNA degradation Add RNase inhibitor; minimize time between dissociation and loading Microglia activation Warm digestion Process at 4 degrees C where possible; actinomycin D has been reported to suppress transcriptional stress responses during digestion in some protocols #### Singulator Protocol Singulator 100/200 -- Mouse Brain Cell Isolation protocol Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Mouse Brain Cell Reagent (papain-based) Auto-Mince : Yes Protocol type : Standard Cell Isolation (1 incubation + 1 grind cycle) Incubation : ~60 minutes at 37 degrees C Single-Shot : 3 mL brain cell reagent + 6 mL DMEM Post-processing : Centrifuge 300g x 5 min at 4 degrees C, RBC lysis if needed, Percoll myelin removal, count with AO/PI Results : Viable cells suitable for scRNA-seq on 10x Genomics, Parse Biosciences, and other platforms Source : Precision Cell Systems Cell Isolation Protocol Guide (100-249-015) #### References [1] Denisenko et al. (2020). Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. Genome Biology 21, 130. [2] 10x Genomics (2023). Demonstrated Protocol: Dissociation of Mouse Tissues for Single Cell RNA Sequencing. CG000183. [3] Worthington Biochemical (2023). Tissue Dissociation Guide. https://www.worthington-biochem.com/tissuedissociation/ [4] Adam M, Potter AS, Potter SS. (2017). Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: A molecular atlas of kidney development. Development 144(19): 3625-3632. DOI: 10.1242/dev.151142. [5] protocols.io: Brain Tissue Dissociation for Cell Sorting. Grant Lin, Michelle Monje (Stanford). https://www.protocols.io/view/brain-tissue-dissociation-for-cell-sorting-nx7dfrn [6] Worthington Papain Dissociation System protocol. Catalog LK003150. [7] Miltenyi Biotec (2023). Adult Brain Dissociation Kit protocol. 130-107-677. [8] Wu et al. (2017). Detecting Activated Cell Populations Using Single-Cell RNA-Seq. Neuron 96(2): 313-329. ### Breast (Normal and Tumor) #### Overview Breast tissue consists of epithelial ducts and lobules embedded in dense fibrous stroma (collagen I and III) and adipose tissue. Dissociation must penetrate this dense ECM while preserving the diverse cell populations including luminal epithelial cells, basal/myoepithelial cells, fibroblasts, adipocytes, immune cells, and endothelial cells. Breast tumors are particularly heterogeneous and may contain regions of necrosis, calcification, or dense desmoplastic stroma that resist enzymatic digestion. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase III 300 U/mL Worthington LS004182 Preferred for breast due to less tryptic activity Collagenase I 1-2 mg/mL Sigma C0130 Alternative; more aggressive Hyaluronidase 100-300 U/mL Sigma H3506 Breaks down hyaluronic acid in stroma DNase I 50-100 U/mL Roche 04716728001 Prevents clumping Trypsin-EDTA 0.05% Gibco 25300054 Brief terminal digest for stubborn clumps (1-2 min only) DMEM/F12 1x Gibco 11330032 Base medium with growth factors FBS 5-10% Various Enzyme inhibition and cell protection ACK Lysis Buffer 1x Gibco A1049201 RBC removal #### Step-by-Step Protocol - Tissue preparation (15 min): Place tissue on ice immediately after excision. Remove fat and necrotic regions. Mince to 1-2 mm pieces with crossed scalpels in a Petri dish with cold DMEM/F12. - Enzymatic digestion (1-3 hours): Incubate in Collagenase III (300 U/mL) + Hyaluronidase (100 U/mL) + DNase I (100 U/mL) in DMEM/F12 + 5% FBS at 37 degrees C with rotation (180 rpm). Monitor every 30 min. - Sequential filtration (10 min): Centrifuge at 300g for 5 min. Resuspend pellet and filter through 100 um, then 70 um, then 40 um cell strainers sequentially. - RBC lysis (5 min): Resuspend in 2 mL ACK buffer, incubate 3-5 min at RT. Quench with 10 mL DMEM/F12. - Dead cell removal (optional, 15 min): Use MACS Dead Cell Removal Kit (Miltenyi 130-090-101) to improve viability before sequencing. - Counting (5 min): Count with AO/PI. Adjust to desired concentration for downstream application. #### Expected Results - Cell yield : 500,000-5,000,000 cells per gram of tissue (highly variable depending on cellularity and fibrosis) - Viability : 70-90% (reduction mammoplasty tissue typically gives higher viability than tumor) - Common cell types recovered : Luminal epithelial, basal/myoepithelial, fibroblasts, adipocytes (low recovery), immune cells (T cells, macrophages, B cells), endothelial cells - Processing time : 2-4 hours total #### Tips and Tricks - Tumor heterogeneity : Sample multiple regions of the tumor to capture spatial heterogeneity. Necrotic cores yield mostly dead cells. - Fat removal is important : Adipose tissue tends to float and can clog strainers. Thorough trimming before mincing typically improves results. - Do not over-digest : Breast epithelial cells are particularly sensitive to prolonged enzymatic treatment. Check dissociation progress at 60 min by examining aliquots under the microscope. - Hormone receptor preservation : For studies requiring intact ER/PR/HER2 surface markers, keep digestion time under 90 min and avoid trypsin. - FACS vs. Flowmi comparison : UC Irvine demonstrated comparable results using FACS versus the Flowmi cell strainer for breast tissue scRNA-seq, with Flowmi offering faster processing. #### Troubleshooting Problem Possible Cause Solution Low viability from tumor Necrotic tissue Trim necrotic regions aggressively; use dead cell removal kit Large clumps persist Dense fibrosis Extend digestion 30 min; increase collagenase concentration Low epithelial cell yield Enzyme too aggressive Switch from Collagenase I to Collagenase III High adipocyte contamination Incomplete fat removal Better physical trimming; Percoll gradient at 1.04 g/mL Cell surface marker loss Over-trypsinization Eliminate trypsin step; use TrypLE Express briefly if needed #### Singulator Protocol Singulator 100/200 -- Pan Tumor Reagent protocol (for breast tumor) Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Pan Tumor Reagent (100-247-099) for tumor; custom enzyme load for normal breast Auto-Mince : Yes Protocol type : Mouse Lung Cell Isolation (2 incubation + 2 grind cycles, the more intensive protocol) Incubation : 37 degrees C, dual-cycle Post-processing : Centrifuge 300g x 5 min, ACK lysis, Percoll debris removal, filter through 40 um strainer Notes : For normal breast tissue, researchers may load their own collagenase + hyaluronidase cocktail via the open reagent platform Source : Precision Cell Systems Pan Tumor Application Note #### References [1] protocols.io: Dissociation of Single Cell Suspensions from Human Breast Tissues. Nicholas Navin (MD Anderson). https://www.protocols.io/view/dissociation-of-single-cell-suspensions-from-human-t3geqjw [2] protocols.io: Breast tumours dissociation. Princess Margaret Cancer Centre. https://www.protocols.io/view/breast-tumours-dissociation-7m9hk96 [3] protocols.io: Human breast tissue dissociation and FACS vs Flowmi for scRNA-Seq. UC Irvine. https://www.protocols.io/view/human-breast-tissue-dissociation-and-facs-vs-flowm-bu2qnydw ### C. elegans #### Overview C. elegans is a model organism with a fully mapped cell lineage (959 somatic cells in adult hermaphrodites). Single-cell dissociation is used for transcriptomic profiling, cell-type-specific studies, and functional genomics. The tough collagenous cuticle of adults and the chitinous eggshell of embryos are the primary barriers to dissociation. Embryos released by bleaching gravid adults are the most common starting material. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Chitinase 1 U/mL Sigma Eggshell degradation Pronase E 15 mg/mL Sigma Cell dissociation SDS-DTT 200 mM DTT + 0.25% SDS -- Cuticle softening (adults/larvae) Collagenase IV 1 mg/mL Sigma Cuticle collagen degradation L-15 medium -- Gibco C. elegans cell culture medium Bleach/NaOH 20% bleach + 0.5 M NaOH -- Embryo isolation from gravid adults Egg buffer 118 mM NaCl, 48 mM KCl -- Isotonic buffer for C. elegans #### Step-by-Step Protocol (Embryos) - Embryo isolation (15 min): Bleach gravid adult worms with alkaline hypochlorite (20% bleach + 0.5 M NaOH) for 5 minutes. Wash embryos 3x in egg buffer. - Eggshell removal (20 min): Treat embryos with Chitinase (1 U/mL) in egg buffer at room temperature for 15-20 minutes until eggshells weaken. - Mechanical + enzymatic dissociation (20 min): Add Pronase E (15 mg/mL) and pipette vigorously through a 25-gauge needle or use Dounce homogenization. - Enzyme neutralization (2 min): Add L-15 medium + 10% FBS. - Filtration (5 min): Filter through 20 um nylon mesh (C. elegans cells are small, 5-10 um). - Wash and count (10 min): Centrifuge 800g x 5 min, resuspend in L-15 + 10% FBS, count. #### Expected Results - Cell yield : 1-5 x 10^6 cells from embryos obtained from one large plate (~10,000 gravid adults) - Viability : 80-95% for embryonic cells - Common cell types recovered : Hypodermal cells, body wall muscle, pharyngeal muscle, neurons, intestinal cells, seam cells, coelomocytes, germ cells #### Tips and Tricks - Embryo staging : Mixed-stage embryos provide the broadest cell type diversity. For specific cell types, synchronize embryo age. - Adult/larval dissociation : The cuticle of post-embryonic stages requires SDS-DTT treatment (200 mM DTT + 0.25% SDS at 37 degrees C for 5 minutes) before enzymatic digestion. This step is critical and cannot be skipped. - Small cell size : C. elegans cells are among the smallest used in single-cell workflows (5-10 um). Verify compatibility with your downstream platform. - Previous challenges on Singulator : A UC Berkeley group reported that C. elegans "did not work at all" on an earlier attempt, suggesting that protocol optimization is needed for this organism. #### Troubleshooting Problem Likely Cause Solution No single cells obtained Cuticle intact (adults) Must use SDS-DTT pre-treatment before enzymatic digestion Low embryo yield from bleach Under-bleaching Extend bleach to 7 min; check bleach freshness Cell clumping DNA from lysed cells Add DNase I (100 U/mL) during pronase step Cells too small for platform C. elegans cell size (5-10 um) Verify platform compatibility; consider nuclei instead Gut granule autofluorescence Intestinal lysosomal granules Gate out in FACS; use glo-1 mutant strain #### Singulator Protocol C. elegans processing on the Singulator platform is an emerging application. Protocol optimization is in early stages. The key challenge is the chitinous eggshell/collagenous cuticle, which requires chemical pre-treatment (SDS-DTT or chitinase) before the Singulator's mechanical + enzymatic processing would be effective. This is an emerging application with no validated Singulator protocol at this time. #### References [1] Literature-standard C. elegans dissociation protocol (Packer et al., 2019, Science; Zhang et al., 2011). ### Cochlea / Inner Ear #### Overview The cochlea contains the organ of Corti with sensory hair cells (inner and outer), supporting cells (Deiters cells, pillar cells, Hensen cells), spiral ganglion neurons, stria vascularis, and various epithelial and mesenchymal cells. Hair cells are extremely rare and fragile. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Thermolysin 0.5 mg/mL Sigma T7902 For separating epithelium Trypsin-EDTA 0.05% Gibco Gentle dissociation Collagenase IV 0.5 mg/mL Worthington For sensory epithelium DNase I 50 U/mL Roche Anti-clumping Accutase 1x Innovative Cell Technologies Gentle alternative to trypsin #### Step-by-Step Protocol - Tissue preparation (15-30 min): Dissect cochlea from temporal bone under microscope. Separate organ of Corti from modiolus and lateral wall. This requires considerable skill. - Enzymatic digestion (20-30 min): Thermolysin (0.5 mg/mL) at 37 degrees C for 10 min, then Trypsin-EDTA (0.05%) or Accutase for 10-15 min with gentle trituration. - Quench and filter (5 min): Add FBS. Filter through 40 um strainer. - Counting (5 min): Count -- expect low total numbers. #### Expected Results - Cell yield : Very low; 1,000-10,000 cells per cochlea for mouse - Viability : 70-90% - Common cell types recovered : Supporting cells (majority), inner hair cells (rare), outer hair cells (extremely rare and fragile), spiral ganglion neurons (if included), strial cells #### Singulator Protocol The cochlea yields very few cells and requires microsurgical dissection that is not compatible with the Singulator's automated tissue loading. Manual protocols are standard. #### References [1] protocols.io: Adult mouse ear dissociation (on ice). Andrew Potter (CCHMC). https://www.protocols.io/view/adult-mouse-ear-dissociation-on-ice-ucwesxe ### Colon / Intestine #### Overview The gastrointestinal tract has a complex layered architecture: mucosa (epithelium + lamina propria), submucosa, muscularis, and serosa. Intestinal epithelial cells turn over every 3-5 days and include absorptive enterocytes, goblet cells, Paneth cells, enteroendocrine cells, and stem cells in the crypts of Lieberkuhn. The lamina propria is rich in immune cells. Dissociation protocols must balance epithelial cell recovery (which requires EDTA-based chelation to release cells from the basement membrane) with immune cell recovery (which requires collagenase-based enzymatic digestion). Mucus removal is a critical preparatory step. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase II 0.5-2 mg/mL Sigma C6885 Primary enzyme for lamina propria cells Collagenase IV 0.5 mg/mL Worthington LS004188 Gentler alternative for epithelial cells EDTA 5-10 mM Sigma E5134 Chelates Ca2+ to release epithelial cells from basement membrane DTT (Dithiothreitol) 1 mM Sigma D9779 Breaks disulfide bonds in mucus layer DNase I 50-100 U/mL Roche Prevents clumping HBSS (Ca/Mg-free) 1x Gibco 14170112 For EDTA chelation steps RPMI 1640 1x Gibco 11875093 Culture medium for enzymatic digestion FBS 2-5% Various Cell protection Percoll 40/80% layers GE Healthcare 17-0891-01 Immune cell enrichment from lamina propria #### Step-by-Step Protocol Combined Epithelial + Immune Cell Protocol : - Tissue preparation (10 min): Place fresh tissue in cold PBS immediately. Flush intestinal lumen with cold PBS using syringe/pipette tip 3x to remove luminal contents. Cut tissue longitudinally and wash with 15 mL cold PBS, agitating to remove mucus. Repeat 2-3x. - Mucus removal (15 min): Incubate tissue pieces in HBSS + 1 mM DTT + 5 mM EDTA at RT for 15 min with gentle agitation. This releases mucus and loosens epithelial cells. - Epithelial cell release (20 min): Transfer tissue to fresh HBSS + 5 mM EDTA (Ca/Mg-free). Shake vigorously at 37 degrees C for 20 min. Collect supernatant (epithelial fraction). Repeat 1-2x for maximum epithelial yield. - Enzymatic digestion of remaining tissue (30-45 min): Mince remaining tissue into 2-3 mm pieces. Digest in RPMI + 2% FBS + Collagenase II (1 mg/mL) + DNase I (100 U/mL) at 37 degrees C with shaking. This releases lamina propria immune cells and stromal cells. - Filtration (5 min): Filter both fractions through 70 um cell strainer. Pool if desired or keep separate for epithelial vs. immune analysis. - Percoll enrichment (optional, 20 min): For immune cell enrichment, layer over 40/80% Percoll gradient. Centrifuge 1000g for 20 min, brake off. Collect interface cells. - Counting (5 min): Count via AO/PI or trypan blue. Adjust concentration. #### Expected Results - Cell yield : 1-10 million cells per cm of intestine (variable by region; ileum > colon for immune cells) - Viability : 80-95% for immune cells; 70-85% for epithelial cells - Common cell types recovered : Enterocytes, goblet cells, Paneth cells, crypt stem cells, enteroendocrine cells, T cells (CD4+, CD8+, gamma-delta), B cells, plasma cells, macrophages, dendritic cells, ILCs, fibroblasts, myofibroblasts, endothelial cells, smooth muscle cells, enteric neurons - Processing time : 60-90 minutes total; ~45 minutes on Singulator #### Tips and Tricks - Mucus removal is make-or-break : Insufficient mucus removal leads to cell clumps, clogged strainers, and contaminated preps. The DTT + EDTA pre-wash is not optional. - EDTA vs. enzyme for epithelial cells : EDTA chelation is gentler than enzymatic digestion for preserving epithelial surface markers, but collagenase is required for deeper lamina propria cells. The two-step approach captures both. - Colon biopsies : For small biopsies (1-3 mm), skip the EDTA pre-treatment and proceed directly to collagenase digestion. Yield will be lower but protocol is faster. - Jejunum requires extra mucus removal : The jejunum has particularly heavy mucus. Consider 2-3 rounds of DTT/EDTA washing. - Cold ischemia time : Intestinal tissue degrades rapidly. Process within 30 min of excision or place immediately in cold HBSS on ice for transport (up to 2 hours). #### Troubleshooting Problem Possible Cause Solution Heavy mucus contamination Insufficient pre-wash Increase DTT to 2 mM; extend pre-wash to 20 min Low epithelial yield Short EDTA treatment Extend chelation to 30 min; increase EDTA to 10 mM Immune cells lost Epithelial fraction not separated Use two-step EDTA then collagenase protocol Cell clumping Insufficient DNase I Increase DNase I to 200 U/mL Poor viability of epithelial cells Mechanical damage Reduce trituration force; use wide-bore pipette tips #### Singulator Protocol Singulator 100/200 -- Intestine Cell Isolation Protocol V2 (official Singulator intestine/colon protocol) Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Intestine Cell Reagent Buffer (3 mL) + DMEM (6 mL) via Single-Shot Tissue prep : Flush 10-12 cm intestine with 10 mL cold PBS 3x, cut lengthwise, wash with 15 mL PBS 2-3x, cut into 2-3 mm pieces, incubate with 15 mL Tissue Pre-Treatment Buffer for 5 min at RT, rinse through 100 um strainer Auto-Mince : Yes Protocol type : Intestine Cell Isolation Protocol V2 Incubation : ~45 minutes at 37 degrees C Post-processing : Centrifuge 300g x 5 min at 4 degrees C, ACK lysis (1 mL, 3-4 min on ice), Percoll debris removal (optional), count with AO/PI on Cellometer K2, strain through 30 um pluriSelect strainer if needed Results : Viable single-cell suspension suitable for scRNA-seq and other downstream applications Source : Precision Cell Systems official Singulator intestine/colon protocol Protocol #### References [1] Precision Cell Systems. Singulator Demonstrated Protocol: Cell Isolation from Intestine Tissue for Single Cell Sequencing. [2] protocols.io: Human colon tissue dissociation for immune cells. Kylie James (Sanger). https://www.protocols.io/view/human-colon-tissue-dissociation-for-immune-cells-tbfeijn [3] protocols.io: Epithelial/Immune Dissociation for Human Colon Biopsies. Broad Institute. https://www.protocols.io/view/epithelial-immune-dissociation-for-human-colon-bio-32igqce [4] protocols.io: Dissociation of fresh colorectal biopsies. Vanderbilt. https://www.protocols.io/view/dissociation-of-fresh-colorectal-biopsies-bci4iugw [5] protocols.io: Dissociation of Jejunum cells for clumps sorting. Weizmann Institute. https://www.protocols.io/view/dissociation-of-jejunum-cells-for-clumps-sorting-bvq3n5yn ### Cornea #### Overview The cornea has five layers: epithelium, Bowman's layer, stroma (keratocytes in collagen lamellae), Descemet's membrane, and endothelium. Corneal dissociation must handle the extremely dense stromal collagen while preserving the delicate endothelial monolayer. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase A 1-2 mg/mL Roche 10103586001 For stromal digestion Trypsin-EDTA 0.25% Gibco For epithelial cells Dispase II 2.4 U/mL Roche Separates epithelium from stroma DNase I 50 U/mL Roche Anti-clumping #### Step-by-Step Protocol - Tissue preparation (5 min): Trephine cornea from globe. For layer-specific analysis, use Dispase II overnight to separate epithelium, then scrape endothelium from Descemet's membrane. - Enzymatic digestion (30-60 min): Collagenase A (1 mg/mL) + DNase I (50 U/mL) for stroma at 37 degrees C; Trypsin-EDTA for epithelium. - Filtration and counting (10 min): Filter through 70 um strainer. Count with AO/PI. #### Expected Results - Cell yield : Low; highly variable by layer and species - Viability : 80-90% - Common cell types recovered : Corneal epithelial cells, keratocytes/stromal fibroblasts, corneal endothelial cells, limbal stem cells (at periphery), immune cells (DCs, macrophages) #### Singulator Protocol Corneal tissue is typically small and processed by hand. The Singulator could process pooled corneal tissue using custom enzyme cocktails if sufficient mass (>20 mg) is available. #### References [1] Collin J, et al. (2021). A single cell atlas of human cornea that defines its development, limbal progenitor cells and their interactions with the immune cells. The Ocular Surface 21: 279-298. ### Dental Pulp #### Overview Dental pulp is the soft connective tissue within the tooth containing odontoblasts, dental pulp stem cells (DPSCs), fibroblasts, endothelial cells, nerve fibers, and immune cells. Access requires tooth extraction and mechanical opening of the pulp chamber. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase I 3 mg/mL Sigma C0130 For dense pulp tissue Dispase II 4 mg/mL Roche Complementary enzyme DNase I 50 U/mL Roche Anti-clumping alpha-MEM 1x Various Standard DPSC culture medium #### Step-by-Step Protocol - Tissue preparation (10 min): Extract tooth. Split open with dental bur or bone rongeurs. Remove pulp with barbed broach or fine forceps. Mince in cold medium. - Enzymatic digestion (45-60 min): Collagenase I (3 mg/mL) + Dispase II (4 mg/mL) + DNase I (50 U/mL) at 37 degrees C. - Filtration and counting (10 min): Filter through 70 um strainer. Count with AO/PI. #### Expected Results - Cell yield : 10,000-500,000 cells per tooth (highly variable with tooth size and patient age) - Viability : 80-95% - Common cell types recovered : DPSCs (Stro-1+, CD146+), fibroblasts, odontoblasts, endothelial cells, pericytes, immune cells, Schwann cells #### Singulator Protocol Dental pulp mass is typically very small ( #### References [1] Gronthos et al. (2000). Postnatal human dental pulp stem cells (DPSCs) in vitro and in vivo. PNAS 97(25): 13625-13630. ### Dorsal Root Ganglia / Peripheral Nerve #### Overview Dorsal root ganglia (DRG) contain sensory neuron cell bodies surrounded by satellite glial cells, Schwann cells, fibroblasts, endothelial cells, and immune cells. Peripheral nerves contain axons wrapped in myelin (Schwann cells), endoneurial fibroblasts, perineurial cells, and blood vessels. Both tissues require gentle dissociation to preserve neuronal viability. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Papain 20 U/mL Worthington LK003150 For neural tissue Collagenase IV 1 mg/mL Worthington LS004188 Alternative for DRG Dispase II 2.4 U/mL Roche Aids in perineurial digestion DNase I 100 U/mL Roche Critical for neural tissue Ovomucoid inhibitor 10 mg/mL Worthington Papain neutralization #### Step-by-Step Protocol - Tissue preparation (15 min): Dissect DRGs from spinal column under microscope. Remove epineurium from peripheral nerve. Collect in cold HBSS. - Enzymatic digestion (30-45 min): Papain (20 U/mL) or Collagenase IV (1 mg/mL) + Dispase II (2.4 U/mL) + DNase I (100 U/mL) at 37 degrees C. - Gentle trituration (5 min): Use fire-polished pipettes with decreasing bore sizes. - Filtration (3 min): Filter through 70 um strainer. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 5,000-50,000 cells per DRG; variable for peripheral nerve segments - Viability : 75-90% - Common cell types recovered : Sensory neurons (various subtypes), satellite glial cells, Schwann cells, fibroblasts, endothelial cells, macrophages #### Singulator Protocol DRG and peripheral nerve samples are typically very small. The Singulator could process pooled DRG tissue or peripheral nerve biopsies using custom papain or collagenase cocktails. The Singulator platform supports peripheral nerve tissue processing with custom enzyme protocols. #### References [1] protocols.io: Ganglia dissociation and single-cell sorting. Columbia. https://www.protocols.io/view/ganglia-dissociation-and-single-cell-sorting-b62frgbn ### Drosophila #### Overview Drosophila is a premier genetic model organism. Single-cell dissociation of embryos, larval tissues (imaginal discs, brains, gut), and adult tissues enables mapping of cell types and gene regulatory networks. Key challenges include the chitinous cuticle of older stages and the small tissue size. Embryos are most commonly dissociated for single-cell RNA-seq atlas projects. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Trypsin 0.5% Gibco Embryo dissociation Collagenase I 2 mg/mL Worthington Larval/adult tissue Papain 14 U/mL Worthington Gentle neural dissociation DNase I 100 U/mL Roche Prevent clumping Schneider's medium -- Gibco Drosophila-specific medium PBS without Ca/Mg -- -- Wash buffer Bleach (sodium hypochlorite) 50% v/v -- Dechorionation BSA 0.04% -- Prevent cell adhesion #### Step-by-Step Protocol (Embryos) - Dechorionation (5 min): Immerse embryos in 50% bleach for 2-3 minutes, rinse thoroughly with water. - Mechanical disruption (5 min): Dounce homogenize or pipette-triturate dechorionated embryos in PBS. - Enzymatic digestion (15-20 min): Add Trypsin (0.5%) or Collagenase I (2 mg/mL) at 25 degrees C (room temperature -- Drosophila physiological range). - Trituration (5 min): Pipette gently every 5 minutes during digestion. - Enzyme neutralization (2 min): Add Schneider's medium + 10% FBS or soybean trypsin inhibitor. - Filtration (5 min): Filter through 40 um cell strainer. For larval brains, use 30 um. - Wash and count (10 min): Centrifuge 300g x 5 min, resuspend in PBS + 0.04% BSA. #### Expected Results - Cell yield : 200,000-500,000 cells from 300-500 embryos; 10,000-50,000 cells per larval brain - Viability : 80-90% for embryos; 70-85% for larval tissues - Common cell types recovered : Variable by stage -- epithelial, mesodermal, neuroblasts, hemocytes, fat body cells, muscle precursors, gut enterocytes, imaginal disc cells #### Tips and Tricks - Room temperature digestion : Drosophila cells are adapted to 25 degrees C. Do NOT heat to 37 degrees C. - Stage timing : Embryonic stage 14-16 (10-13 hours after egg laying) provides the broadest cell type diversity for atlas projects. - Cuticle challenge : Third instar larvae and adults have tough cuticles. Dissect target tissues (brains, imaginal discs, guts) out before enzymatic digestion rather than trying to dissociate intact animals. - Hemocyte contamination : Drosophila hemocytes (blood cells) are abundant and can dominate scRNA-seq libraries. Consider depletion by differential adhesion. #### Troubleshooting Problem Likely Cause Solution Cuticle fragments in prep Intact cuticle pieces Increase filtration stringency; pre-dissect tissue from cuticle Low viability Temperature too high Keep at room temperature (25C) throughout Hemocyte dominance in scRNA-seq Natural abundance of blood cells Pre-plate on glass for 30 min to deplete adherent hemocytes Incomplete embryo dissociation Bleach residue inhibiting enzymes Rinse 5+ times after dechorionation Yolk contamination Early embryos Use late-stage embryos (>10 hours AEL) For Drosophila embryo nuclei isolation, the standard nuclei isolation protocol with the Nuclei Isolation Cartridge (blue) at Slow disruption speed is the recommended starting point. Researchers have successfully used this approach for 300-500 embryo batches, following published manual protocols (Calderon et al. 2022, DOI: 10.1126/science.abn5800). For cell isolation, Trypsin or Collagenase I loaded via Single-Shot using the Cell Isolation Cartridge (white) is suggested, but the instrument should NOT be set to 37 degrees C -- room temperature operation is essential. #### References [1] Li H, et al. (2022). Fly Cell Atlas: A single-nucleus transcriptomic atlas of the adult fruit fly. Science. DOI: 10.1126/science.abk2432. Calderon D, et al. (2022). The continuum of Drosophila embryonic development at single-cell resolution. Science. DOI: 10.1126/science.abn5800. ### Endometrium / Uterus #### Overview The endometrium is the inner mucosal lining of the uterus, consisting of a functionalis layer (shed during menstruation) and a basalis layer (regenerative). It contains luminal and glandular epithelial cells, stromal fibroblasts, endothelial cells, smooth muscle cells (in the myometrium), and various immune cells (uterine NK cells, macrophages, T cells). The ECM composition and tissue architecture change dramatically across the menstrual cycle, requiring protocol adaptation based on cycle phase. Collagenase-based or trypsin-based protocols are both commonly used. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase V 0.5-1 mg/mL Sigma C9263 Standard for endometrium Collagenase IV 0.5 mg/mL Worthington LS004188 Alternative Trypsin-EDTA 0.25% Gibco Alternative single-enzyme approach DNase I 50-100 U/mL Roche Anti-clumping RPMI or DMEM 1x Various Base medium FBS 10% Various Enzyme quenching #### Step-by-Step Protocol Collagenase Protocol (Sanger/HCA) : - Tissue preparation (10 min): Place fresh endometrial biopsy or curetting on ice. Remove blood clots and mucus. Mince into 1-2 mm pieces. - Enzymatic digestion (60 min): Incubate in Collagenase V (0.5 mg/mL) + DNase I (100 U/mL) in RPMI + 10% FBS at 37 degrees C with agitation (200 rpm). Triturate every 15 min. - Filtration (5 min): Filter through 70 um cell strainer. Wash with cold RPMI. - RBC lysis (if needed, 5 min): ACK buffer for 3-5 min. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : Variable depending on biopsy size and cycle phase; 100,000-5,000,000 cells - Viability : 80-95% - Common cell types recovered : Luminal epithelial cells, glandular epithelial cells, stromal fibroblasts, uterine NK cells, macrophages, T cells, endothelial cells, smooth muscle cells (if myometrium included) - Processing time : 75-90 minutes #### Tips and Tricks - Menstrual cycle phase matters : Proliferative phase tissue is easier to dissociate than secretory phase, which has more ECM. - Myometrium requires separate processing : If studying both layers, separate endometrium from myometrium before dissociation. Myometrium is much denser and requires longer digestion. - Endometriosis tissue : Ectopic endometrial lesions are highly fibrotic and require extended digestion (up to 90 min) or higher enzyme concentrations. #### Troubleshooting Problem Possible Cause Solution Low yield from biopsy Small sample size Reduce volumes proportionally; optimize enzyme concentrations Mucus contamination Insufficient pre-wash Wash tissue 3-5 times before mincing Low stromal cell yield Short digestion Extend to 75-90 min #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : User-supplied collagenase via Single-Shot mechanism Notes : The Singulator Standard Nuclei protocol protocol (pig endometrium) demonstrates compatibility with endometrial tissue for nuclei isolation. For cell isolation, use the open reagent platform with collagenase cocktail. The Single-Shot delivers 3 mL enzyme + 6 mL buffer. Source : Precision Cell Systems. Singulator Demonstrated Protocol: Nuclei Isolation and Cleanup from Frozen Tissue (Standard protocol). #### References [1] protocols.io: Endometrium dissociation with collagenase. Sanger Institute. https://www.protocols.io/view/endometrium-dissociation-with-collagenase-76thren [2] protocols.io: Human endometrium and endometriosis tissue dissociation for scRNA-seq. Jackson Lab. https://www.protocols.io/view/human-endometrium-and-endometriosis-tissue-dissoci-bvy8n7zw [3] protocols.io: Myometrium Single Cell Dissociation Protocol. U Michigan. https://www.protocols.io/view/myometrium-single-cell-dissociation-protocol-bmbek2je ### Esophagus #### Overview The esophagus is lined by a stratified squamous epithelium that is continuously renewed from basal stem/progenitor cells. Below the epithelium lies the lamina propria with immune cells and fibroblasts, the muscularis mucosae, submucosa, and muscularis propria. Dissociation protocols must access both the epithelial compartment and the subepithelial immune/stromal populations. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 0.5 mg/mL Worthington LS004188 For stromal/immune cells Trypsin-EDTA 0.25% Gibco For epithelial cell release DNase I 50-100 U/mL Roche Anti-clumping HBSS 1x Various Base buffer #### Step-by-Step Protocol - Tissue preparation (10 min): Separate epithelium from muscularis if desired. Mince epithelial tissue into 1-2 mm pieces. - Epithelial dissociation (30-45 min): Incubate in Trypsin-EDTA (0.25%) + DNase I (50 U/mL) at 37 degrees C with shaking. - Quench and filter (5 min): Add FBS to 10%. Filter through 70 um cell strainer. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : Variable; 500,000-2,000,000 cells per cm squared of epithelium - Viability : 80-90% - Common cell types recovered : Basal keratinocytes, suprabasal keratinocytes, fibroblasts, immune cells, endothelial cells - Processing time : 45-60 minutes #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : User-supplied trypsin or collagenase via Single-Shot #### References [1] protocols.io: CGAP Human Oesophagus Epithelium Dissociation. CGAP. https://www.protocols.io/view/cgap-human-oesophagus-epithelium-dissociation-qz8dx9w ### Heart #### Overview Cardiac tissue is densely packed with cardiomyocytes (which can be 100-150 um in length), fibroblasts, endothelial cells, smooth muscle cells, pericytes, immune cells (macrophages, T cells), and neuronal cells. Cardiomyocytes are exceptionally fragile due to their large size and rod-shaped morphology. Collagenase-based digestion is standard, but the large cell size means that standard microfluidic-based scRNA-seq platforms (10x Chromium, which has an upper limit of ~40 um) cannot capture intact cardiomyocytes -- specialized platforms like Parse Biosciences (fixation-based) or single-nucleus approaches are preferred for cardiomyocyte representation. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase II 1-2 mg/mL Worthington LS004176 Standard for cardiac dissociation Collagenase IV 0.5-1 mg/mL Worthington LS004188 Gentler alternative DNase I 50-100 U/mL Roche Prevents clumping CaCl2 1 mM Various Required for collagenase activity on cardiac tissue Trypsin 0.05% Gibco Brief final digest for stubborn clumps only RBC Lysis Buffer 1x G-Biosciences Heart is highly vascularized DMEM or HBSS 1x Various Base medium #### Step-by-Step Protocol - Tissue preparation (10 min): Excise heart. Perfuse briefly with cold PBS to remove blood. Cut into 4 roughly symmetrical pieces. Wash away blood thoroughly. - Mincing (5 min): Mince 100-200 mg of tissue into 1 mm pieces with fine scissors in cold HBSS + DNase I (50 U/mL). - Enzymatic digestion (30-45 min): Incubate in Collagenase II (1.5 mg/mL) + CaCl2 (1 mM) + DNase I (50 U/mL) in HBSS at 37 degrees C with gentle rocking. Triturate gently every 10 min with wide-bore pipette. - Sequential digestion (optional, 15-20 min per round): Remove supernatant containing released cells. Add fresh enzyme to remaining tissue. Repeat 2-3 times for maximum yield. - Filtration (5 min): Filter through 100 um strainer (to capture large cardiomyocytes) or 70 um strainer (to exclude them). Choice depends on whether cardiomyocytes are desired. - RBC lysis (5 min): Resuspend in 1 mL RBC Lysis Buffer for 3-5 min on ice. Quench with 8 mL DMEM. - Counting (5 min): Count with AO/PI. Note that cardiomyocytes will be a small fraction of total cells in droplet-based platforms. #### Expected Results - Cell yield : ~23,000 cells per mg of tissue (mouse heart, Singulator) - Viability : 68% overall; higher for non-cardiomyocyte populations - Common cell types recovered : Endothelial cells (~39%), fibroblasts (~22%), macrophages/DCs (~10%), pericytes (~7%), smooth muscle cells (~5%), endocardial cells (~3-4%), cardiomyocytes (~3-4%), T cells (~3%), B cells (~3%), lymphatic endothelial cells, erythrocytes, epithelial cells, neuronal cells - Processing time : 45-60 minutes total; ~30 minutes on Singulator #### Tips and Tricks - Cardiomyocyte capture requires special cartridges : On the Singulator, use the Large Cell Isolation Cartridge (red top, 100-258-668) which has wider filter apertures to preserve large cardiomyocytes. - Parse Biosciences is preferred for cardiomyocyte representation : The fixation-based Evercode platform captures cells regardless of size. The Precision Cell Systems application note demonstrated 3-4% cardiomyocyte representation using Parse Biosciences WT v2. - Perfusion is strongly recommended : Residual blood in cardiac tissue can significantly inflate cell counts with erythrocytes and obscure downstream data. Thorough perfusion before dissociation is standard practice. - CaCl2 is generally required : Collagenases typically require calcium for activity. Avoid using Ca-free HBSS during enzymatic digestion steps. - Gentle trituration only : Cardiomyocytes are easily damaged by pipetting. Use cut pipette tips (wide bore) and minimal trituration force. #### Troubleshooting Problem Possible Cause Solution Very low cardiomyocyte yield Cell size exclusion by strainer Use 100 um or no strainer; use Large Cell Isolation Cartridge High erythrocyte contamination Incomplete perfusion Perfuse more thoroughly; extend ACK lysis to 5 min Low overall viability Over-digestion Reduce enzyme time to 25-30 min Clumping DNase I insufficient Increase to 100 U/mL; add fresh DNase I during trituration Poor sequencing quality from cardiomyocytes Droplet platform size limit Switch to Parse Biosciences or single-nucleus approach #### Singulator Protocol Singulator 100/200 -- Mouse Heart Cell Isolation protocol Cartridge : Large Cell Isolation Cartridge (100-258-668, red top) Reagent : Mouse Heart Cell Isolation Reagent (100-253-846) Auto-Mince : Yes Sample : ~150 mg fresh mouse heart tissue, cut into 4 pieces, blood washed away Protocol type : Standard Cell Isolation (1 incubation + 1 grind cycle) Incubation : 37 degrees C Post-processing : ACK RBC lysis, count with AO/PI on Nexcelom K2 Results : ~23,000 cells/mg at 68% viability; 10,264 cells analyzed via scRNA-seq showing 13 distinct cell types including cardiomyocytes (3.2%) and endocardial cells (3.5%) Source : Precision Cell Systems Application Note: Isolation of Viable Cells from Mouse Heart Tissue with the Singulator 100 (P/N: 100-261-728) #### References [1] Precision Cell Systems Application Note: Isolation of Viable Cells from Mouse Heart Tissue with the Singulator 100. P/N: 100-261-728. [2] protocols.io: JAX-Sen: Mouse heart dissociation for scRNA-seq. Jackson Lab. https://www.protocols.io/view/jax-sen-mouse-heart-dissociation-for-single-cell-r-dgkw3uxe ### iPSC-Derived Tissues #### Overview Induced pluripotent stem cell (iPSC)-derived tissues and standard 2D cell cultures represent a distinct category where dissociation is typically simpler than primary tissue. iPSC-derived cardiomyocytes, neurons, hepatocytes, and other differentiated cell types may be grown as monolayers or 3D aggregates. The primary challenge is maintaining cell type identity and viability during dissociation, particularly for post-mitotic cells like cardiomyocytes and neurons that are sensitive to enzymatic treatment. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Accutase 1x STEMCELL Technologies iPSC and neuron dissociation TrypLE Express 1x Gibco General 2D culture dissociation Trypsin-EDTA 0.05% Gibco Standard monolayer detachment Collagenase IV 1 mg/mL Worthington Colony lifting (iPSCs) DNase I 50 U/mL Roche Prevent clumping Y-27632 10 uM Various iPSC survival after dissociation EDTA 0.5 mM -- Gentle iPSC colony dissociation PBS without Ca/Mg -- -- Wash and EDTA buffer #### Step-by-Step Protocol (iPSC-Derived Cells) - Medium removal (2 min): Aspirate culture medium. Rinse once with PBS without Ca/Mg. - Enzymatic dissociation (5-10 min): Add Accutase (1x) for neurons/iPSCs or TrypLE Express for other cell types. Incubate at 37 degrees C. - Gentle detachment (3 min): Tap flask/plate to detach cells. Gently pipette to dissociate colonies into single cells. - Neutralization (2 min): Add 3 volumes of culture medium. For trypsin, add medium + 10% FBS. - Collection (5 min): Centrifuge 200-300g x 3-5 min. Resuspend in appropriate medium + Y-27632 for iPSCs. - Count (5 min): Count with AO/PI or trypan blue. Assess for single-cell suspension. #### Expected Results - Cell yield : Variable by flask/plate size; typically > 90% recovery from monolayers - Viability : 90-98% for monolayer cultures; 80-90% for iPSC-derived differentiated cells - Common cell types : iPSCs, iPSC-derived cardiomyocytes, iPSC-derived neurons, iPSC-derived hepatocytes, iPSC-derived epithelial cells, and all standard cell lines #### Tips and Tricks - iPSC fragility : iPSCs and iPSC-derived neurons are highly sensitive to trypsin. Use Accutase or 0.5 mM EDTA in PBS for the gentlest dissociation. - ROCK inhibitor for iPSCs : Y-27632 (10 uM) should be added to the collection medium to prevent dissociation-induced death of iPSCs and iPSC-derived cardiomyocytes. - 3D iPSC aggregates : iPSC-derived embryoid bodies (EBs) or cardiac aggregates require longer digestion (15-20 min) and may benefit from collagenase pre-treatment. - EDTA method for iPSC colonies : For passaging iPSC colonies without full singularization, 0.5 mM EDTA in PBS for 3-5 minutes at room temperature releases colonies as small clumps rather than single cells. #### Troubleshooting Problem Likely Cause Solution iPSC death after passaging No ROCK inhibitor Add Y-27632 (10 uM) to collection medium Incomplete detachment Insufficient enzyme time Extend to 10-15 min; check enzyme freshness Cell clumps Incomplete dissociation of colonies Pipette more vigorously; use Accutase + DNase I Loss of pluripotency markers Over-exposure to trypsin Switch to Accutase or EDTA method Cardiomyocyte death Trypsin sensitivity Use TrypLE Express (gentler) or Collagenase IV #### Singulator Protocol While iPSC-derived tissues and 2D cultures do not represent the Singulator's primary use case (solid tissue dissociation), the platform supports these applications. For iPSC-derived 3D aggregates or thick multilayer cultures, the Singulator provides standardized dissociation using Accutase or TrypLE loaded via the Single-Shot mechanism with the Cell Isolation Cartridge (white). Protocols for both nuclei and cell isolation from plated cultures have been validated. For standard 2D monolayers, traditional flask-based enzymatic detachment is generally simpler and more appropriate than using the Singulator. #### References [1] Literature-standard iPSC and cell culture dissociation protocols (Beers et al., 2012; Braam et al., 2010; STEMCELL Technologies iPSC protocols). [2] protocols.io: Dissociation of EBs using Worthington Kit. NYSCF. https://www.protocols.io/view/dissociation-of-ebs-using-worthington-kit-dftv3nn6 ### Kidney #### Overview The kidney has a highly organized architecture with the cortex (containing glomeruli, proximal and distal tubules, and collecting ducts) and the medulla (loops of Henle and collecting ducts). The diversity of cell types -- podocytes, proximal tubule cells, distal tubule cells, collecting duct cells (intercalated and principal), thick ascending limb cells, endothelial cells, mesangial cells, fibroblasts, and various immune cells -- makes kidney dissociation both valuable and challenging. Proximal tubule cells are particularly abundant and relatively easy to recover, while podocytes and endothelial cells require gentler conditions. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1-2 mg/mL Worthington LS004188 Standard for kidney tubular dissociation Collagenase II 0.5-1 mg/mL Sigma C6885 Alternative for glomerular-enriched preps Liberase TL 0.1-0.25 mg/mL Roche 05401020001 For gentle dissociation preserving podocytes DNase I 50-100 U/mL Roche Prevents clumping Hyaluronidase 100 U/mL Sigma H3506 Optional; helps with medullary tissue HBSS or RPMI 1x Various Base buffer BSA 0.04-1% Various Cell protection during processing ACK Lysis Buffer 1x Various RBC removal (kidney is highly vascularized) #### Step-by-Step Protocol - Tissue preparation (10 min): Decapsulate kidney. For regional analysis, separate cortex from medulla. Mince into 1-2 mm pieces on ice in cold HBSS. - Enzymatic digestion (30-45 min): Incubate in Collagenase IV (1 mg/mL) + DNase I (100 U/mL) in RPMI or HBSS at 37 degrees C with gentle agitation (80-100 rpm on orbital shaker). Triturate every 10-15 min. - Filtration (5 min): Pass through 100 um then 70 um then 40 um cell strainers sequentially. Wash each strainer with cold HBSS. - RBC lysis (5 min): Resuspend in 2-3 mL ACK buffer for 3-5 min on ice. Quench with 10 mL medium. - Debris removal (optional, 15 min): Percoll gradient (35-45%) for removal of debris and dead cells. - Wash and count (5 min): Centrifuge 300g x 5 min, resuspend in cold HBSS + 0.04% BSA. Count with AO/PI. #### Expected Results - Cell yield : ~35,600 cells per mg (mouse, Singulator); highly variable for human biopsy tissue - Viability : 85-95% (mouse); 70-85% (human) depending on cold ischemia time - Common cell types recovered : Proximal tubule cells (dominant), distal tubule cells, thick ascending limb cells, collecting duct principal and intercalated cells, podocytes (low yield), endothelial cells, mesangial cells, fibroblasts, macrophages, T cells, B cells - Processing time : ~25 min on Singulator + 15 min wash/RBC lysis; 45-60 min manual #### Tips and Tricks - Cold on-ice protocol : Andrew Potter (CCHMC) developed a cold dissociation protocol that processes kidney entirely at 4 degrees C, reducing transcriptional artifacts for scRNA-seq at the cost of somewhat lower yield. - Tubule-dominant preps : Kidney dissociations are dominated by proximal tubule cells (often 40-60% of total). To enrich for rarer cell types, consider FACS sorting or magnetic enrichment post-dissociation. - Human kidney biopsies : For small needle biopsies, reduce enzyme volume proportionally and shorten digestion time to 20-30 min. - Kidney is a moderate RNase activity tissue : RNase inhibitor is recommended but not as critical as for lung or pancreas. - Tumor vs. normal kidney : Kidney tumors (RCC) can be processed with Pan Tumor Reagent or Collagenase IV at higher concentration (2 mg/mL). #### Troubleshooting Problem Possible Cause Solution Proximal tubule cells dominate Normal tissue composition Use FACS/MACS enrichment for cell types of interest Low podocyte yield Fragile cells lost during processing Reduce mechanical force; use Liberase TL instead of collagenase High debris Medullary tissue inclusion Separate cortex from medulla before processing Low viability Extended cold ischemia Process tissue within 30 min of excision; keep on ice Cell clumping Insufficient DNase Increase DNase I to 150 U/mL #### Singulator Protocol Singulator 100/200 -- Mouse Kidney Cell Isolation protocol Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Mouse Kidney Cell Reagent (100-064-631) Auto-Mince : Yes Sample : ~220 mg fresh tissue, finely minced Protocol type : Standard Cell Isolation (1 incubation + 1 grind cycle) Incubation : ~25 minutes on instrument + 15 min post-processing Single-Shot : 3 mL kidney cell reagent + 6 mL DMEM or HBSS Post-processing : Centrifuge 300g x 5 min at 4 degrees C, ACK RBC lysis 3-5 min, resuspend in 1 mL DMEM/HBSS + 1% BSA, count with AO/PI on Nexcelom K2 Results : ~35,600 cells/mg at 88.5% viability; 18,417 kidney cells captured via Parse Biosciences showing 12 cell types Source : Precision Cell Systems Application Note: Sequencing Skin and Kidney Cells (P/N: 100-261-065) #### References [1] Precision Cell Systems Application Note: Sequencing Skin and Kidney Cells: Leveraging the Singulator 100 and Parse Biosciences' Evercode WT v2 Platform. P/N: 100-261-065. [2] protocols.io: Adult human kidney tissue cell dissociation (on ice). Andrew Potter (U Cincinnati). https://www.protocols.io/view/adult-human-kidney-tissue-cell-dissociation-on-ice-q6cdzaw [3] protocols.io: JAX-Sen: Mouse kidney dissociation for scRNA-seq. Jackson Lab. https://www.protocols.io/view/jax-sen-mouse-kidney-dissociation-for-single-cell-dgkv3uw6 [4] protocols.io: Human Kidney Tumor Dissociation for single-cell genomics. Univ Rennes/Inserm. https://www.protocols.io/view/human-kidney-tumor-dissociation-for-single-cell-g-2n2gdge ### Liver #### Overview The liver is the largest solid organ, composed primarily of hepatocytes (~80% of liver volume) arranged in lobules with sinusoidal endothelial cells, Kupffer cells (resident macrophages), hepatic stellate cells (Ito cells), cholangiocytes (bile duct epithelial cells), and various immune cells. Hepatocytes are large (20-40 um), binucleated, metabolically active cells that are extremely sensitive to enzymatic and mechanical damage. Traditional liver dissociation via collagenase perfusion yields high-quality hepatocytes, but for scRNA-seq from tissue pieces (rather than whole organ perfusion), alternative approaches are needed. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 0.5-1 mg/mL Worthington LS004188 Standard for liver tissue pieces Collagenase P 0.5 mg/mL Roche 11213857001 Gentler alternative Liberase TM 0.1-0.25 mg/mL Roche 05401127001 Thermolysin + Collagenase; good for NPCs DNase I 50-100 U/mL Roche Prevents clumping Hyaluronidase 50-100 U/mL Sigma H3506 Optional; aids in perisinusoidal matrix HBSS with Ca/Mg 1x Various Calcium required for collagenase activity ACK Lysis Buffer 1x Various Critical -- liver is blood-rich Percoll 25-50% gradient Various Hepatocyte purification by density FBS 2-5% Various Enzyme quenching and cell protection #### Step-by-Step Protocol From Tissue Pieces (Not Perfusion) : - Tissue preparation (10 min): Place fresh liver tissue on ice immediately. Remove Glisson's capsule if intact. Mince into 1-2 mm pieces in cold HBSS. Wash 2-3 times with cold HBSS to remove blood. - Enzymatic digestion (20-30 min): Incubate in Collagenase IV (0.5-1 mg/mL) + DNase I (100 U/mL) in HBSS with Ca/Mg at 37 degrees C with gentle agitation. Triturate every 10 min. - Filtration (5 min): Filter through 100 um cell strainer. Wash with cold HBSS + 2% FBS. - Low-speed centrifugation (5 min): Centrifuge at 50g for 3 min to pellet hepatocytes. Supernatant contains non-parenchymal cells (NPCs). This differential centrifugation is the key to separating hepatocytes from NPCs. - NPC enrichment (5 min): Centrifuge the NPC-containing supernatant at 300g for 5 min. - RBC lysis (5 min): Resuspend each fraction in ACK buffer for 3-5 min. Quench with HBSS. - Optional Percoll cleanup (15 min): For hepatocyte purification, resuspend in 25% Percoll and centrifuge at 100g for 5 min. Live hepatocytes pellet; dead cells float. - Counting (5 min): Count hepatocytes and NPCs separately with AO/PI. #### Expected Results - Cell yield : ~30,000 cells/mg total (Singulator); NPCs are the dominant fraction - Viability : >80% total; hepatocyte viability specifically is 60-70% (they are inherently fragile) - Common cell types recovered : Hepatocytes (lower proportional yield from non-perfusion protocols), sinusoidal endothelial cells (LSEC), Kupffer cells, stellate cells, cholangiocytes, NK/NKT cells, T cells, B cells - Processing time : 40-60 minutes manual; ~25 min on Singulator + 15 min post-processing #### Tips and Tricks - Non-parenchymal cells dominate non-perfusion preps : When dissociating from tissue pieces (rather than whole-organ collagenase perfusion), expect 60-80% of recovered cells to be non-parenchymal. This is a known limitation of non-perfusion methods. - Hepatocyte viability is inherently lower : Hepatocytes are large and metabolically fragile. Viability of 60-70% for hepatocytes specifically is considered acceptable. - Differential centrifugation is strongly recommended : The 50g spin separates large hepatocytes from small NPCs. Omitting this step typically results in mixed cell populations that complicate downstream analysis. - Liver has moderate-high RNase activity : Add RNase inhibitor for scRNA-seq applications. - Human biopsy considerations : Human liver biopsies from surgical resections have variable cold ischemia times. Process as quickly as possible and note ischemia time for QC. #### Troubleshooting Problem Possible Cause Solution Very few hepatocytes recovered Non-perfusion method limitation Accept NPC-enriched prep, or use whole-organ perfusion protocol Low hepatocyte viability Mechanical damage Reduce trituration; use cut tips; minimize pipetting Heavy blood contamination Insufficient washing Wash tissue 3-5 times before mincing; extend ACK lysis Cell clumping Dead hepatocytes releasing DNA Increase DNase I; use fresh enzyme preparation Poor scRNA-seq data from hepatocytes RNA degradation in these metabolically active cells Add RNase inhibitor; process rapidly; consider single-nucleus approach #### Singulator Protocol Singulator 100/200 -- Mouse Liver Cell Isolation protocol Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Mouse Liver Cell Reagent Auto-Mince : Yes Protocol type : Standard Cell Isolation (1 incubation + 1 grind cycle) Incubation : ~25 minutes on instrument Post-processing : Centrifuge 300g x 5 min at 4 degrees C, ACK RBC lysis, count with AO/PI Results : ~30,000 cells/mg with >80% total viability; hepatocyte-specific viability of 60-70%; preferentially isolates non-parenchymal cells (60-80% NPC) Notes : The Singulator was noted to preferentially isolate non-parenchymal cells from liver. For hepatocyte-enriched preps, differential centrifugation (50g) post-Singulator processing is recommended Source : Precision Cell Systems; Protocol Guide #### References [1] protocols.io: Human Liver Tissue Dissociation for 10x Single Cell RNA-seq. Ankur Sharma (GIS). https://www.protocols.io/view/human-liver-tissue-dissociation-for-10x-single-cel-vhue36w [2] protocols.io: Human Liver Caudate Lobe Dissociation for ScRNA-seq v2. Sonya MacParland (U Toronto). https://www.protocols.io/view/human-liver-caudate-lobe-dissociation-for-scrna-se-m9sc96e ### Lung (Normal and Tumor) #### Overview Lung tissue is composed of more than 40 distinct cell types organized across conducting airways (bronchi, bronchioles) and gas exchange regions (alveoli). The alveolar region contains type I and type II pneumocytes, while airways contain ciliated cells, club cells, goblet cells, basal cells, and neuroendocrine cells. The lung interstitium contains fibroblasts, smooth muscle cells, and a rich vasculature with endothelial cells. Immune cells are abundant, including alveolar macrophages, interstitial macrophages, dendritic cells, T cells, B cells, NK cells, and mast cells. The lung ECM contains collagen I, III, IV, elastin, and proteoglycans. Liberase TH or similar collagenase/thermolysin blends are the most commonly validated enzymes for lung dissociation. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Liberase TH 1 mg/mL in RPMI Roche 05401151001 Collagenase I/II + thermolysin; validated for lung scRNA-seq Collagenase I 2 mg/mL Sigma C0130 Alternative; more variable than Liberase Elastase 4 U/mL Worthington LS002279 Optional; helps with elastic fibers in alveoli DNase I 50-100 U/mL Roche Prevents clumping (critical for lung) RPMI 1640 1x Gibco 11875093 Preferred base medium for lung HBSS 1x Gibco Alternative base buffer FBS 10% Various For quenching enzymes ACK Lysis Buffer 1x Various RBC removal (lung is highly vascularized) RNase Inhibitor 1 U/uL Sigma Protector Critical -- lung has high RNase activity #### Step-by-Step Protocol Standard Protocol (37 degrees C) : - Tissue preparation (10 min): Place fresh lung tissue on ice immediately. For mouse, perfuse via right ventricle with cold PBS to clear blood. Trim off visible airways and blood vessels for peripheral lung enrichment if desired. Mince into 1-2 mm pieces. - Enzymatic digestion (30-45 min): Incubate in Liberase TH (1 mg/mL) + DNase I (100 U/mL) in RPMI at 37 degrees C with gentle agitation (120 rpm orbital shaker). Triturate gently every 10 min. Lung tissue should visibly dissociate after 20-30 min. - Enzyme quenching (2 min): Add equal volume of cold RPMI + 10% FBS to stop digestion. - Filtration (5 min): Filter through 70 um cell strainer. Wash with cold RPMI. - RBC lysis (5 min): Centrifuge 300g x 5 min. Resuspend in 2-3 mL ACK buffer for 3-5 min at RT. Quench with 10 mL RPMI. - Counting and QC (5 min): Count with AO/PI. Assess viability. Target >80% viability for scRNA-seq. Cold (On-Ice) Protocol -- Andrew Potter, CCHMC: - All steps performed at 4 degrees C to minimize transcriptional artifacts - Liberase TL (cold-active formulation) used instead of Liberase TH - Extended digestion time (45-60 min) compensates for reduced enzyme activity at 4 degrees C - Yields are lower but RNA quality is superior for scRNA-seq #### Expected Results - Normal lung cell yield : 645,000-2,635,000 cells per sample (82-96% viability) across 3 patients - Tumor lung cell yield : 2,787,000-6,732,000 cells per sample (90-99% viability) across 3 patients - Viability : Normal lung 82-96%; tumor 76-99% - Common cell types recovered : Type I and II pneumocytes, ciliated cells, club cells, basal cells, fibroblasts, smooth muscle cells, endothelial cells (lymphatic and vascular), alveolar macrophages, interstitial macrophages, DCs, T cells (CD4+, CD8+), B cells, NK cells, mast cells, neutrophils - Processing time : ~45 minutes on Singulator; 60-90 minutes manual #### Tips and Tricks - RNase inhibitor is non-negotiable : Lung tissue has very high RNase expression. Add RNase inhibitor to enzyme solutions, wash buffers, and resuspension media. 10x Genomics specifically recommends this for lung samples. - Liberase TH vs. Collagenase + Elastase : Liberase TH at 1 mg/mL in RPMI is the validated standard for Singulator lung protocols. This is NOT the same as "Collagenase + Elastase" -- do not substitute without validation. - Mouse Lung Cell Isolation protocol has dual incubation : Unlike other Singulator cell protocols, the lung protocol runs two incubation and two grind cycles because lung tissue requires more aggressive processing. - Tumor vs. normal uses different reagent : Normal lung uses Mouse Lung Cell Reagent (100-064-413); tumors use Pan Tumor Reagent (100-247-099). Both use the Lung Cell Isolation protocol on the Singulator. - TIL isolation compatible : The Singulator preserves CD45 surface epitopes, enabling downstream TIL purification via EasySep CD45+ positive selection after dissociation. - Reproducibility : Precision Cell Systems demonstrated Pearson correlations of 0.936 or higher for average gene expression across 3 patient normal lung preps processed on the Singulator. - Protocol optimization : The Mouse Lung Cell Isolation protocol can be shortened to 2 x 10 min incubations with 1 mg/mL Liberase using cell cartridges for fixed tissue dissociation. #### Troubleshooting Problem Possible Cause Solution Low viability Over-digestion; RNase degradation Reduce enzyme time; ensure RNase inhibitor present Shorter cDNA fragment size (10x) May reflect tumor biology rather than degradation per se Confirm by running +/- RNase inhibitor comparison; shorter fragments observed in lung tumors even with quality RNA Heavy RBC contamination Incomplete perfusion (mouse) or vascularized tissue Perfuse mouse lung with PBS via right ventricle; extend ACK lysis Low epithelial cell yield Enzyme insufficient Increase Liberase TH to 1.5 mg/mL; extend digestion to 45 min Ambient RNA contamination Cell lysis during processing Run SoupX on sequencing data; minimize processing time; keep cold Poor reproducibility Manual variation Use Singulator for standardized processing #### Singulator Protocol Singulator 100/200 -- Mouse Lung Cell Isolation protocol Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Mouse Lung Reagent (100-064-413) for normal; Pan Tumor Reagent (100-247-099) for tumor Auto-Mince : Yes Protocol type : Mouse Lung Cell Isolation (2 incubation + 2 grind cycles) Incubation : ~45 minutes at 37 degrees C Single-Shot : 3 mL enzyme + 6 mL DMEM/RPMI Post-processing : Centrifuge 300g x 5 min at 4 degrees C, ACK RBC lysis 5 min, Percoll density gradient debris removal, filter, count with AO/PI Results : Normal: 645K-2.6M cells, 82-96% viability. Tumor: 2.8M-6.7M cells, 76-99% viability. scRNA-seq: 6,000-8,500 cells per library with broad cell type representation Reproducibility : Pearson correlation >= 0.936 across 3 patient normal lung preps Source : Precision Cell Systems Application Note: Single-Cell Sequencing of Tumor-Infiltrating Lymphocytes in Lung Cancer; Application Note: Dissociating Tumor Tissue into Cells with Pan Tumor Reagent #### References [1] Guilliams et al. (2022). Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. Cell 185(2): 379-396. [2] Precision Cell Systems Application Note: Single-Cell Sequencing of Tumor-Infiltrating Lymphocytes in Lung Cancer: A Comparative Analysis Using the Singulator Platform. [3] Precision Cell Systems Application Note: Dissociating Tumor Tissue into Cells with Precision Cell Systems' Pan Tumor Reagent. P/N: 100-261-619. [4] protocols.io: Adult mouse lung cell dissociation (on ice) v4. Andrew Potter (CCHMC). https://www.protocols.io/view/adult-mouse-lung-cell-dissociation-on-ice-cfiytkfw [5] protocols.io: Adult human lung cell dissociation (on ice) v2. Andrew Potter (CCHMC). https://www.protocols.io/view/adult-human-lung-cell-dissociation-on-ice-ch6dt9a6 [6] protocols.io: Single Cell Dissociation of Fresh Lung Tissue. Lance Peter, Mei-I Chung (TGen). https://www.protocols.io/view/single-cell-dissociation-of-fresh-lung-tissue-7xkhpkw [7] protocols.io: Cell dissociation of fresh human lung tissue for scRNA-seq. Ilias Angelidis (Helmholtz Munich). https://www.protocols.io/view/cell-dissociation-of-fresh-human-lung-tissue-for-s-zp2f5qe ### Lymph Node #### Overview Lymph nodes are encapsulated organs containing organized lymphoid tissue with distinct cortical (B cell follicles), paracortical (T cell zones), and medullary regions. Like spleen, lymph nodes are soft and relatively easy to dissociate mechanically. The capsule and trabecular framework contain collagen-rich stromal cells that require enzymatic digestion for recovery. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase D 1 mg/mL Roche 11088858001 Preserves immune cell markers DNase I 50 U/mL Roche Anti-clumping RPMI 1x Gibco Base medium FBS 2-5% Various Cell protection #### Step-by-Step Protocol - Mechanical dissociation (5-10 min): Place lymph node on a 70 um cell strainer over a 50 mL tube. Gently press with syringe plunger while washing with cold RPMI + 2% FBS. This releases >90% of lymphocytes. - Optional enzymatic digestion (20-30 min): For stromal/DC recovery, mince remaining tissue and digest in Collagenase D (1 mg/mL) + DNase I (50 U/mL) at 37 degrees C. - Filtration (5 min): Filter through 40 um strainer. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 5-50 million cells per mouse lymph node; human inguinal lymph nodes can yield 100+ million - Viability : >95% - Common cell types recovered : B cells (germinal center, memory, naive), T cells (CD4+, CD8+, Tregs), follicular DCs, plasmacytoid DCs, macrophages, fibroblastic reticular cells (with enzyme), high endothelial venule cells #### Singulator Protocol Lymph node typically does not require the Singulator platform due to its soft, easily dissociated nature. Mechanical pressing through a cell strainer is sufficient for most applications. #### References [1] Fletcher AL, Acton SE, Knoblich K. (2015). Lymph node fibroblastic reticular cells in health and disease. Nature Reviews Immunology 15(6): 350-361. ### Mammary Gland #### Overview The mammary gland shares many features with breast tissue (see Breast section) but is specifically studied in developmental biology and lactation research. Mouse mammary gland dissociation is a common model system. The tissue contains a bilayered epithelium (luminal and myoepithelial cells) embedded in adipose and collagenous stroma. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase I 2 mg/mL Sigma C0130 Standard for mammary Trypsin 0.05% Gibco Gentle secondary digest DNase I 50 U/mL Roche Anti-clumping DMEM/F12 1x Gibco Base medium Dispase 5 U/mL StemCell Technologies For organoid dissociation #### Step-by-Step Protocol - Tissue preparation (10 min): Dissect mammary fat pads (#3, #4 for mouse). Remove lymph node. Mince into 1 mm pieces. - Enzymatic digestion (1-2 hours): Incubate in Collagenase I (2 mg/mL) + Trypsin (0.05%) + DNase I (50 U/mL) in DMEM/F12 at 37 degrees C with shaking (125 rpm). - Differential centrifugation (15 min): Pulse-centrifuge at 350g for 10 sec to pellet epithelial organoids. Remove fat and single cells in supernatant. Repeat 3-5 times. - Organoid dissociation (10 min): To obtain single cells, incubate organoids in Trypsin-EDTA (0.25%) + DNase I at 37 degrees C for 5-10 min with pipetting. - Filtration and counting (10 min): Filter through 40 um strainer. Count with AO/PI. #### Expected Results - Cell yield : 1-10 million cells per mouse mammary gland - Viability : 85-95% - Common cell types recovered : Luminal cells (hormone-sensing, secretory), basal/myoepithelial cells, stromal cells, immune cells #### Singulator Protocol Singulator 100/200 -- Custom enzyme via open reagent platform. The Singulator can handle mammary tissue using collagenase cocktails via the Single-Shot mechanism. #### References [1] Shackleton et al. (2006). Generation of a functional mammary gland from a single stem cell. Nature 439: 84-88. ### Muscle (Skeletal) #### Overview Skeletal muscle is composed of multinucleated myofibers (mature muscle cells), satellite cells (muscle stem cells residing beneath the basal lamina), fibroblasts/fibro-adipogenic progenitors (FAPs), endothelial cells, pericytes, Schwann cells, and immune cells. The dense collagenous endomysium, perimysium, and epimysium surrounding muscle fibers make dissociation challenging. Myofibers are extremely large (>100 um diameter, centimeters in length) and cannot be captured on standard scRNA-seq platforms; single-nucleus RNA-seq is preferred for myofiber profiling. For mononuclear cell isolation, the protocol must efficiently digest the ECM while preserving delicate satellite cells. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase B 1-2 mg/mL Roche 11088815001 Primary enzyme for muscle ECM Dispase II 2.4-4.8 U/mL Roche 04942078001 Complementary enzyme; cleaves fibronectin and collagen IV DNase I 50-100 U/mL Roche Prevents clumping from necrotic fibers Collagenase II 0.2-0.5% (w/v) Worthington LS004176 Alternative primary enzyme HBSS or DMEM 1x Various Base buffer FBS 10-20% Various Enzyme quenching and cell protection #### Step-by-Step Protocol - Tissue preparation (10 min): Dissect skeletal muscle (e.g., hindlimb muscles from mouse). Remove tendon, fat, and connective tissue. Mince finely into 1-2 mm pieces with scissors in cold HBSS. - First enzyme digest (30 min): Incubate in Collagenase B (2 mg/mL) + DNase I (50 U/mL) in DMEM at 37 degrees C with shaking (180 rpm). Triturate every 10 min. - Second enzyme digest (15-20 min): Add Dispase II (2.4 U/mL) to the same tube. Continue incubation at 37 degrees C. Triturate every 5 min until no visible fragments remain. - Filtration (5 min): Filter through 70 um then 40 um cell strainers. Wash with DMEM + 10% FBS. - RBC lysis (if needed, 5 min): ACK buffer for 3-5 min on ice. - Wash and count (5 min): Centrifuge 300g x 5 min. Resuspend in cold HBSS + 2% FBS. Count with AO/PI. #### Expected Results - Cell yield : Highly variable; 100,000-1,000,000 mononuclear cells per gram depending on muscle type and species - Viability : 80-95% for mononuclear cells - Common cell types recovered : Satellite cells (Pax7+), FAPs (Pdgfra+), endothelial cells, pericytes, Schwann cells, macrophages, T cells. Mature myofibers are NOT recovered as intact cells - Processing time : 60-75 minutes total #### Tips and Tricks - Collagenase B + Dispase II is the standard muscle combination : This is different from most other tissues. Do not substitute Collagenase I or IV without validation. - Two-step enzyme digestion : The sequential addition of Dispase II after Collagenase B is more effective than adding both simultaneously. - Satellite cell enrichment : For satellite cell studies, sort for CD34+/Sca1-/CD45-/CD31- (mouse) or CD56+/CD45- (human) after dissociation. - Injury models : Injured or regenerating muscle yields more satellite cells and immune cells. Reduce digestion time for injured tissue to preserve activated cell populations. #### Troubleshooting Problem Possible Cause Solution Low satellite cell yield Insufficient ECM digestion Increase Collagenase B to 2 mg/mL; extend first digest to 40 min Myofiber contamination Incomplete filtration Use 40 um strainer to exclude large fragments Low viability Over-digestion with Dispase Reduce Dispase II time to 10 min Cell clumping DNA from damaged myofibers Increase DNase I to 100 U/mL #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : User-supplied Collagenase B + Dispase II via Single-Shot mechanism Auto-Mince : Yes Protocol type : Standard Cell Isolation or custom parameters Notes : Skeletal muscle is not among the pre-formulated Precision Cell Systems reagents, but the open reagent platform supports loading custom enzyme cocktails. A validated protocol using Collagenase B + Dispase II has been demonstrated. Source : Precision Cell Systems open reagent platform documentation #### References [1] Liu et al. (2015). Isolation of skeletal muscle stem cells by fluorescence-activated cell sorting. Nature Protocols 10, 1612-1624. ### Organoids and Spheroids #### Overview Organoids and spheroids are three-dimensional (3D) cell culture systems that recapitulate aspects of tissue architecture. Dissociation into single cells is required for passaging, single-cell genomics, and functional assays. Organoids vary dramatically in their matrix embedding (Matrigel, BME, collagen) and tissue of origin, which dictates the dissociation approach. Brain organoids, intestinal organoids, and tumor spheroids are the most common types processed to single cells. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose TrypLE Express 1x Gibco Gentle trypsin alternative Accutase 1x STEMCELL Technologies Stem cell-compatible dissociation Dispase II 1 mg/mL Roche Matrigel digestion Cell Recovery Solution 1x Corning Matrigel dissolution (4C) Trypsin-EDTA 0.05% Gibco Standard dissociation DNase I 50 U/mL Roche Prevent clumping Y-27632 (ROCK inhibitor) 10 uM Various Prevent dissociation-induced apoptosis PBS without Ca/Mg -- -- Wash buffer #### Step-by-Step Protocol (Matrigel-Embedded Organoids) - Matrigel dissolution (30 min): Place organoid culture on ice. Add Cell Recovery Solution or cold PBS and incubate at 4 degrees C for 20-30 minutes until Matrigel dissolves. - Collection (5 min): Gently pipette to release organoids from dissolved matrix. Centrifuge 200g x 3 min at 4 degrees C. - Enzymatic dissociation (10-15 min): Resuspend organoid pellet in pre-warmed TrypLE Express or Accutase at 37 degrees C. - Mechanical disruption (5 min): Pipette every 3-5 minutes with a P1000 tip. For tough organoids, use a 27-gauge needle. - Neutralization (2 min): Add 3 volumes of culture medium + 10% FBS. - Filtration (5 min): Filter through 40 um strainer. - ROCK inhibitor (optional): Resuspend in medium + Y-27632 (10 uM) to improve survival after dissociation. - Wash and count (5 min): Centrifuge 300g x 5 min, resuspend in PBS + 0.04% BSA, count. #### Expected Results - Cell yield : 50,000-500,000 cells per organoid well (highly variable by organoid size and type) - Viability : 75-90% with ROCK inhibitor; 60-80% without - Common cell types recovered : Depends on organoid type -- intestinal (stem cells, enterocytes, goblet cells, Paneth cells), brain (neurons, glia, progenitors), tumor (cancer cells, CAFs if co-cultured) #### Tips and Tricks - ROCK inhibitor is strongly recommended : Organoid cells are commonly sensitive to dissociation-induced anoikis. Y-27632 at 10 uM has been shown to significantly improve survival in most organoid systems. - Matrigel removal first : Incomplete Matrigel dissolution leads to cell clumps that clog microfluidic devices. Allow full 30 minutes at 4 degrees C. - Brain organoids : A Stanford researcher found that nuclei isolation reduced debris compared to cell dissociation, though yield required comparison to manual methods. Flash-frozen mini-brain organoid nuclei were successfully isolated using the standard nuclei protocol with Slow disruption speed. - Young vs. old organoids : Younger organoids dissociate more readily. Older, more mature organoids with increased ECM may need extended TrypLE incubation. - Suspension spheroids : Spheroids not embedded in matrix (e.g., tumor spheroids) can skip the Matrigel dissolution step and go directly to enzymatic digestion. #### Troubleshooting Problem Likely Cause Solution Cell clumps after digestion Matrigel residue or insufficient enzyme Extend cold PBS incubation; increase TrypLE time to 20 min Low viability ( Anoikis after dissociation Add Y-27632 (10 uM) immediately; keep cells cold after enzyme Over-digestion TrypLE too long for small organoids Check every 5 min; small organoids may need only 5-8 min Debris on scRNA-seq Matrigel fragments Use Cell Recovery Solution instead of cold PBS for dissolution Variable yield between wells Heterogeneous organoid sizes Pool multiple wells before dissociation for consistency #### Singulator Protocol Organoid processing on the Singulator has yielded promising results. Testing with young and old organoids for both single cell and nuclei isolation showed key findings: (1) Nuclei isolation reduced debris compared to single-cell dissociation; (2) Single cell dissociation was more challenging with viability at 75% for young samples post-centrifugation; (3) Further optimization may be needed for yield comparison versus manual methods. Flash-frozen mini-brain organoids were successfully processed using the standard nuclei isolation protocol with Slow disruption speed, producing clean nuclei visualized after concentration. For organoid cell isolation, TrypLE Express loaded via Single-Shot (3 mL enzyme + 6 mL buffer) with the Cell Isolation Cartridge (white) is the recommended approach. For nuclei, the standard Nuclei Isolation Cartridge (blue) at Slow disruption provides validated results. #### References [1] Literature-standard organoid dissociation protocols (Sato et al., 2011; Lancaster & Knoblich, 2014; Huch et al., 2015). [2] protocols.io: Single cell dissociation of brain organoids. DZNE Tubingen. https://www.protocols.io/view/single-cell-dissociation-of-brain-organoids-crwsv7ee ### Ovary #### Overview The ovary contains follicles at various stages of maturation (primordial, primary, secondary, antral), corpus luteum, granulosa cells, theca cells, stromal cells, surface epithelial cells, and immune cells. The ovarian cortex is dense and collagen-rich, requiring aggressive enzymatic digestion. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1-2 mg/mL Worthington LS004188 Standard for ovary DNase I 100 U/mL Roche Essential for this tissue Hyaluronidase 100 U/mL Sigma H3506 Aids in follicular matrix HBSS 1x Various Base buffer #### Step-by-Step Protocol - Tissue preparation (10 min): Mince ovary into 1-2 mm pieces on ice. - Enzymatic digestion (30-60 min): Collagenase IV (1 mg/mL) + Hyaluronidase (100 U/mL) + DNase I (100 U/mL) at 37 degrees C with shaking. - Filtration (5 min): Filter through 100 um then 70 um strainers. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : Variable; 500,000-2,000,000 cells per ovary depending on age and species - Viability : 75-90% - Common cell types recovered : Granulosa cells, theca cells, surface epithelial cells, stromal fibroblasts, endothelial cells, immune cells, oocytes (rare in single-cell preps) #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform with Collagenase IV cocktail. #### References [1] protocols.io: Human Fallopian Tube and Ovary Dissociation for scRNA-seq. U Chicago. https://www.protocols.io/view/human-fallopian-tube-and-ovary-dissociation-for-si-bfudjns6 ### Pancreas #### Overview The pancreas contains two functionally distinct compartments: the exocrine pancreas (acinar cells and ductal cells, comprising ~95% of the organ) and the endocrine pancreas (islets of Langerhans with alpha, beta, delta, epsilon, and PP cells, comprising ~1-2%). Acinar cells are extremely rich in digestive enzymes (trypsin, chymotrypsin, lipase, amylase) and RNases, making pancreatic dissociation uniquely challenging -- released enzymes will digest other cells and degrade RNA if not managed aggressively. Additionally, acinar cells are fragile and die rapidly during dissociation, contributing to high ambient RNA levels in sequencing data. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase P 0.5-1 mg/mL Roche 11213857001 Preferred for pancreas due to minimal endotoxin Collagenase V 1 mg/mL Sigma C9263 Alternative with lower tryptic activity DNase I 100-200 U/mL Roche Higher concentration needed due to acinar cell death Soybean Trypsin Inhibitor (STI) 0.1 mg/mL Sigma T9128 Blocks released trypsin from acinar cells RNase Inhibitor 1 U/uL Sigma Protector or SUPERase-In CRITICAL -- pancreas has extremely high RNase activity HBSS or RPMI 1x Various Base buffer BSA 0.1-1% Various Enzyme quenching and surface adsorption protection FBS 10% Various Additional protection #### Step-by-Step Protocol - Tissue preparation (10 min): Place fresh pancreas on ice immediately. Remove fat, lymph nodes, and connective tissue. Mince into 1-2 mm pieces in cold HBSS + STI (0.1 mg/mL) + DNase I (100 U/mL). - Enzymatic digestion (20-30 min): Incubate in Collagenase P (0.5 mg/mL) + DNase I (100 U/mL) + STI (0.1 mg/mL) in HBSS at 37 degrees C with gentle agitation. Monitor carefully -- pancreas digests faster than most tissues. - Enzyme quenching (immediate): Add equal volume ice-cold HBSS + 10% FBS + STI to stop digestion. All subsequent steps at 4 degrees C. - Filtration (5 min): Filter through 70 um then 40 um cell strainers. - Wash (10 min): Centrifuge 300g x 3 min. Wash 2x with cold HBSS + 0.04% BSA + RNase inhibitor. - Counting (5 min): Count immediately with AO/PI. Load onto scRNA-seq platform as rapidly as possible. #### Expected Results - Cell yield : Variable; 500,000-5,000,000 cells per gram - Viability : 60-80% (lower than most tissues due to acinar cell fragility) - Common cell types recovered : Acinar cells (majority, but many die), ductal cells, islet endocrine cells (alpha, beta, delta), stellate cells, endothelial cells, macrophages, T cells - Processing time : 40-60 minutes total #### Tips and Tricks - RNase inhibitor in EVERY buffer : Pancreas is the highest RNase-expressing tissue. Add RNase inhibitor to lysis buffer, wash buffers, and final resuspension medium. Do not skip this step. - Soybean trypsin inhibitor (STI) is strongly recommended : Released trypsin from acinar cells can digest other cell types. STI blocks this activity. SBTI at 0.1 mg/mL is commonly used. - Speed is important : Processing pancreas first in multi-tissue experiments is recommended. Minimizing time from harvest to loading helps preserve RNA quality. - Islet enrichment : For islet-focused studies, use density gradient separation (Histopaque-1077/1119) to enrich endocrine cells away from exocrine debris. - SoupX or CellBender for ambient RNA : Pancreatic scRNA-seq data commonly has high ambient RNA from lysed acinar cells. Post-processing bioinformatic correction is recommended. #### Troubleshooting Problem Possible Cause Solution Very low viability Acinar cell self-digestion Increase STI; reduce digestion time to 15-20 min High ambient RNA in scRNA-seq Acinar cell lysis Run SoupX/CellBender; increase RNase inhibitor; reduce time Low endocrine cell yield Islets embedded in exocrine tissue Use density gradient enrichment; increase digestion time slightly Rapid cell death during processing Released proteases Keep all buffers ice-cold post-digestion; maximize STI Clumping DNA from dead acinar cells Increase DNase I to 200 U/mL #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : User-supplied Collagenase P + DNase I + STI via Single-Shot mechanism, or use Precision Cell Systems reagents as available RNase inhibitor : Add directly to dissociation chamber with sample Notes : Pancreas has high RNase activity. Precision Cell Systems recommends adding RNase inhibitor to the dissociation chamber for all pancreas applications. Source : Precision Cell Systems -- RNase inhibitor recommendations for pancreas #### References [1] protocols.io: Adult mouse pancreas cell dissociation (on ice) v2. Andrew Potter (CCHMC). https://www.protocols.io/view/adult-mouse-pancreas-cell-dissociation-on-ice-cfi3tkgn [2] Baron et al. (2016). A Single-Cell Transcriptomic Map of the Human and Mouse Pancreas. Cell Systems 3(4): 346-360. ### Placenta #### Overview The placenta is a highly vascularized organ at the maternal-fetal interface, containing trophoblast cells (cytotrophoblasts, syncytiotrophoblasts, extravillous trophoblasts), endothelial cells, stromal cells (Hofbauer cells, fibroblasts), and maternal immune cells (decidual NK cells, macrophages, T cells). The extensive villous architecture and fibrinoid deposits require thorough enzymatic digestion. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1 mg/mL Worthington LS004188 Standard Trypsin 0.25% Gibco Brief secondary digest for epithelial cells DNase I 100 U/mL Roche Critical for this blood-rich tissue HBSS 1x Various Base buffer ACK Lysis Buffer 1x Various Extensive RBC contamination from maternal blood Percoll 40/70% gradient Various Trophoblast enrichment #### Step-by-Step Protocol - Tissue preparation (15 min): Dissect villous tissue from the fetal side. Remove decidua and membranes. Wash extensively with cold PBS (5-7 washes) to remove maternal blood. Mince into 1-2 mm pieces. - Enzymatic digestion (30-45 min): Collagenase IV (1 mg/mL) + DNase I (100 U/mL) at 37 degrees C with agitation. - Filtration (5 min): Filter through 100 um then 70 um strainers. - RBC lysis (5-10 min): Multiple rounds of ACK lysis may be needed. - Optional trophoblast enrichment (30 min): Percoll gradient (40/70%) to enrich trophoblasts. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : Highly variable; 1-50 million cells per gram depending on gestational age - Viability : 75-90% - Common cell types recovered : Cytotrophoblasts, extravillous trophoblasts, Hofbauer cells (fetal macrophages), stromal fibroblasts, endothelial cells, decidual NK cells, decidual macrophages, T cells #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform. Placenta is compatible with the Singulator's Cell Isolation Cartridge using custom collagenase cocktails. #### References [1] protocols.io: JAX-Sen: Single Cell Dissociation of Mouse Placenta. Jackson Lab. https://www.protocols.io/view/jax-sen-single-cell-dissociation-of-mouse-placenta-hcnzb2vf7 ### Prostate #### Overview The prostate gland contains acinar epithelial cells (luminal, basal, neuroendocrine), fibromuscular stromal cells, smooth muscle cells, endothelial cells, and immune cells. The stroma is dense with collagen and smooth muscle. Prostate cancer is a major application for single-cell approaches. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase I 1-2 mg/mL Sigma C0130 For stromal/glandular dissociation Collagenase IV 1 mg/mL Worthington Alternative; gentler Hyaluronidase 100 U/mL Sigma H3506 Aids in glandular matrix DNase I 100 U/mL Roche Anti-clumping Trypsin-EDTA 0.05% Gibco Brief terminal digest if needed RPMI 1x Various Base medium #### Step-by-Step Protocol - Tissue preparation (10 min): Place prostate tissue on ice. Remove fat and seminal vesicles. Mince into 1-2 mm pieces. - Enzymatic digestion (60-90 min): Collagenase I (1 mg/mL) + Hyaluronidase (100 U/mL) + DNase I (100 U/mL) in RPMI + 5% FBS at 37 degrees C with rotation. - Optional secondary digest (10 min): Brief Trypsin-EDTA (0.05%) incubation at 37 degrees C for remaining clumps. - Filtration (5 min): Filter through 70 um then 40 um strainers. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 500,000-5,000,000 cells per gram - Viability : 75-90% - Common cell types recovered : Luminal epithelial cells, basal epithelial cells, neuroendocrine cells (rare), fibroblasts, smooth muscle cells, endothelial cells, macrophages, T cells #### Singulator Protocol Singulator 100/200 -- Custom enzyme via open reagent platform, or Pan Tumor Reagent for prostate tumors. #### References [1] Henry et al. (2018). A cellular anatomy of the normal adult human prostate and prostatic urethra. Cell Reports 25(12): 3530-3542. ### Retina / Eye #### Overview The retina is a delicate neural tissue with a highly organized layered structure containing photoreceptors (rods and cones), bipolar cells, ganglion cells, amacrine cells, horizontal cells, Muller glia, microglia, and retinal pigment epithelium (RPE). Dissociation must be extremely gentle to preserve these fragile neural cell types. Papain is the preferred enzyme, similar to brain tissue. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Papain 20 U/mL Worthington LK003150 Most commonly used enzyme for retinal dissociation DNase I 100 U/mL Worthington Anti-clumping Ovomucoid inhibitor 10 mg/mL Worthington Stops papain EBSS or Ames' medium 1x Various Balanced salt solution #### Step-by-Step Protocol - Tissue preparation (10 min): Enucleate eye. Dissect retina from RPE and sclera under a dissecting microscope. Place in cold Ames' medium or EBSS. - Enzymatic digestion (30-45 min): Incubate in activated papain (20 U/mL) + DNase I (100 U/mL) in EBSS at 37 degrees C. Gently triturate every 10 min. - Trituration (5 min): Add ovomucoid inhibitor. Triturate gently with fire-polished Pasteur pipette. - Filtration (3 min): Filter through 40 um strainer. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 100,000-1,000,000 cells per retina depending on species - Viability : 80-95% - Common cell types recovered : Rod photoreceptors (dominant), cone photoreceptors, bipolar cells, ganglion cells, amacrine cells, horizontal cells, Muller glia, microglia, RPE cells (if included) #### Singulator Protocol Retinal tissue is extremely delicate and typically processed by hand with papain dissociation kits under microscope guidance. The Singulator may be too aggressive for intact retinal dissociation but could process pooled retinal tissue for bulk preparations via custom papain loading. #### References [1] protocols.io: Single-Cell Dissociation of Human Trabecular Meshwork. Harvard/Mass Eye and Ear. https://www.protocols.io/view/single-cell-dissociation-of-human-trabecular-meshw-bfdyji7w ### Salivary Gland #### Overview Salivary gland tissue consists of acinar cells (serous and mucous), ductal epithelial cells, myoepithelial cells, and stromal components. The gland's mucin-rich extracellular matrix requires careful enzymatic treatment. Research applications include Sjogren's syndrome, salivary gland tumors, and regenerative medicine for radiation-induced xerostomia. Mouse submandibular glands are the most commonly used model. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Collagenase II 2 mg/mL Worthington ECM degradation Hyaluronidase 1 mg/mL Sigma Mucin matrix breakdown Dispase II 2.4 U/mL Roche Basement membrane digestion DNase I 100 U/mL Roche Prevent clumping CaCl2 2 mM -- Collagenase cofactor BSA 0.1% -- Cell protection HBSS with Ca/Mg -- Gibco Digestion medium #### Step-by-Step Protocol - Tissue preparation (10 min): Excise salivary glands, remove connective tissue capsule, and mince finely with scalpels in cold HBSS. - Primary enzymatic digestion (30 min): Incubate minced tissue in Collagenase II (2 mg/mL) + Hyaluronidase (1 mg/mL) + DNase I (100 U/mL) in HBSS with Ca/Mg at 37 degrees C with gentle agitation. - Mechanical disruption (5 min): Triturate with 1 mL pipette every 10 minutes during digestion. - Secondary digestion (15 min): Add Dispase II (2.4 U/mL) for remaining 15 minutes to release ductal cells. - Filtration (5 min): Filter through 100 um then 40 um strainers to remove acinar clumps. - Wash and count (10 min): Centrifuge 300g x 5 min, resuspend in PBS + 0.04% BSA, count with trypan blue or AO/PI. #### Expected Results - Cell yield : 1-3 x 10^6 cells per mouse submandibular gland - Viability : 80-90% - Common cell types recovered : Acinar cells (serous/mucous), ductal epithelial cells, myoepithelial cells, endothelial cells, fibroblasts, immune cells, nerve-associated cells #### Tips and Tricks - Mucin interference : Salivary glands produce substantial mucin that can trap cells. Hyaluronidase is typically needed to help break down this matrix. - Acinar cell fragility : Serous acinar cells are mechanically fragile. Minimize trituration force and use wide-bore pipette tips. - Cold collection : Keep excised glands on ice immediately to preserve RNA integrity. - Species differences : Human salivary gland tissue may require longer digestion times (45-60 min) compared to mouse tissue. #### Troubleshooting Problem Likely Cause Solution Mucin clumps in suspension Insufficient hyaluronidase Increase hyaluronidase to 1.5 mg/mL or extend digestion Low acinar cell yield Over-digestion Reduce primary digestion to 20 min Poor viability Mechanical damage Use wider-bore pipettes, reduce trituration passes Ductal cell clumps Incomplete Dispase digestion Increase Dispase concentration or duration #### Singulator Protocol Researchers at UCSF have inquired about using the Singulator for mouse salivary gland nuclei isolation. The Singulator community website includes user-submitted protocols for salivary gland tissue. The standard nuclei isolation protocol with the Nuclei Isolation Cartridge (blue) should be applicable. For cell isolation, a custom enzyme cocktail of Collagenase II + Hyaluronidase loaded via the Single-Shot mechanism (3 mL enzyme + 6 mL buffer) would be recommended, using the Cell Isolation Cartridge (white) with its built-in 150 um + 70 um filters. #### References [1] Literature-standard salivary gland dissociation protocol based on Collagenase II + Hyaluronidase approach widely used in salivary gland biology (Emmerson et al., 2017; Lombaert et al., 2017). ### Skin / Dermis #### Overview Skin is a multi-layered organ comprising the epidermis (stratified epithelium with keratinocytes, melanocytes, Langerhans cells, Merkel cells), the dermis (dense connective tissue with fibroblasts, endothelial cells, smooth muscle, nerve endings), and subcutaneous adipose tissue. Hair follicles span multiple layers and contain specialized stem cell populations. The dermal ECM is extremely dense with Collagen I, III, and VII, requiring aggressive enzymatic digestion. Despite this density, the Singulator platform has demonstrated 5,200-5,300 cells/mg with 89.5-90% viability from mouse skin. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1-2 mg/mL Worthington LS004188 Standard for dermal dissociation Liberase TH 0.5-1 mg/mL Roche 05401151001 Alternative with thermolysin activity Trypsin 0.25% Gibco For epidermal separation (dispase-trypsin protocol) Dispase II 2.4 U/mL Roche Separates epidermis from dermis at the basement membrane DNase I 50-100 U/mL Roche Standard anti-clumping DMEM or HBSS 1x Various Base medium FBS 10% Various Enzyme quenching #### Step-by-Step Protocol Combined Epidermal + Dermal Protocol : - Tissue preparation (15 min): Remove subcutaneous fat. For mouse skin, shave or use depilatory cream to remove fur, then excise dorsal skin. Rinse in cold PBS. Trim to desired size. - Optional epidermis-dermis separation (overnight or 1 hour): Float skin dermis-side-down on Dispase II (2.4 U/mL) at 4 degrees C overnight or 37 degrees C for 1 hour. Peel epidermis away with forceps. Process each layer separately or together. - Mincing (5 min): Mince tissue into 1-2 mm pieces. For Singulator processing, mince to ~1-2 mm squared pieces. - Enzymatic digestion (45-60 min): Incubate in Collagenase IV (1 mg/mL) + DNase I (100 U/mL) in DMEM at 37 degrees C with agitation. For Singulator, use the Mouse Skin Cell Isolation protocol. - Filtration (5 min): Filter through 70 um cell strainer. Wash with cold DMEM + 10% FBS. - Wash and count (10 min): Centrifuge 300g x 5 min at 4 degrees C. Resuspend in DMEM. Centrifuge again. Final resuspension in 1 mL DMEM. Count with AO/PI. #### Expected Results - Cell yield : 5,200-5,300 cells/mg (mouse, Singulator) - Viability : 89.5-90% (AO/PI) - Common cell types recovered : Permanent epidermal keratinocytes, anagen hair follicle keratinocytes, fibroblasts, T cells, immune cells, endothelial cells, Schwann cells, melanocytes, skeletal muscle cells - Processing time : 60-90 minutes manual; automated with Singulator - scRNA-seq quality : 2,302 genes per cell, 20,092 mean reads per cell, 92.3% fraction reads in cells, 88% of cells showing #### Tips and Tricks - Skin nuclei are tricky : Skin nuclei isolation is particularly challenging due to the fibrous nature of the tissue. Mince as much as possible and use NIC+ cartridge for nuclei. - Fur removal : For mouse skin, depilatory cream (Nair) for 3-5 minutes is gentler than shaving and reduces razor damage to the epidermis. - Cold ischemia sensitivity : Skin cells tolerate moderate cold ischemia (up to 2 hours on ice) better than most internal organs. - Broad cell type representation demonstrated : scRNA-seq from Singulator-processed skin showed 14 distinct cell types including rare Schwann cells and melanocytes, confirming preservation of fragile cell types. #### Troubleshooting Problem Possible Cause Solution Low yield Dense ECM inadequately digested Extend digestion to 60 min; increase collagenase Hair follicle debris Incomplete fur removal Use depilatory cream thoroughly before excision Keratinocyte clumps Incomplete dissociation of epidermis Add Dispase II pre-treatment; increase trituration Melanocyte loss UV-induced apoptosis during handling Protect from light exposure; work under amber light #### Singulator Protocol Singulator 100/200 -- Mouse Skin Cell Isolation protocol Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Mouse Skin Cell Reagent (100-254-082) Auto-Mince : Yes Sample : ~249 mg fresh tissue, fur removed, finely minced to ~1-2 mm squared Protocol type : Standard Cell Isolation (Mouse Skin Cell Isolation protocol) Incubation : 37 degrees C (single incubation + grind cycle) Post-processing : Centrifuge 300g x 5 min at 4 degrees C, resuspend in 3 mL DMEM, centrifuge again, final resuspension in 1 mL DMEM, count with AO/PI Results : ~5,200 cells/mg at 90% viability; 6,851 cells analyzed via scRNA-seq showing 14 cell types, 2,302 genes/cell, 92.3% reads in cells Source : Precision Cell Systems Application Note: Isolation of Mouse Skin Cells with the Singulator Platform (P/N: 100-261-837) #### References [1] Precision Cell Systems Application Note: Isolation of Mouse Skin Cells with the Singulator Platform. P/N: 100-261-837. [2] protocols.io: Human skin single cell dissociation. Newcastle/Sanger. https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn ### Spinal Cord #### Overview Spinal cord tissue presents unique dissociation challenges due to its high myelin content, dense extracellular matrix, and mixture of neurons, glial cells, and extensive white matter tracts. Cell isolation is important for studying spinal cord injury, ALS, multiple sclerosis, and motor neuron diseases. Both fresh and frozen human spinal cord samples are processed, with papain being the preferred enzyme for neural tissue. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Papain 30 U/mL Worthington Primary dissociation DNase I 100-200 U/mL Roche Prevent clumping EBSS -- Worthington Digestion buffer Ovomucoid inhibitor 10 mg/mL Worthington Papain neutralization Myelin removal beads per manufacturer Miltenyi Myelin depletion Percoll 22%/35% gradient GE Healthcare Debris/myelin removal BSA 0.02% -- Cell protection #### Step-by-Step Protocol - Tissue preparation (15 min): Dissect spinal cord from vertebral column, remove meninges carefully. Mince into 1-2 mm pieces in cold EBSS. - Enzymatic digestion (45 min): Incubate in papain (30 U/mL) + DNase I (200 U/mL) in EBSS at 37 degrees C. - Trituration (5 min): Gently triturate with fire-polished Pasteur pipettes of decreasing bore size. - Papain neutralization (5 min): Add ovomucoid inhibitor solution. - Myelin removal (30 min): Either Percoll gradient centrifugation (22%/35%) or Miltenyi myelin removal beads with MACS columns. - Filtration and wash (10 min): Filter through 70 um strainer, wash in PBS + 0.02% BSA, centrifuge 300g x 5 min. - Counting (5 min): Count with AO/PI to distinguish cells from myelin debris. #### Expected Results - Cell yield : 0.5-2 x 10^6 cells per cm of mouse spinal cord; variable for human tissue - Viability : 70-85% (lower than brain due to white matter processing) - Common cell types recovered : Motor neurons, interneurons, astrocytes, oligodendrocytes, OPCs, microglia, ependymal cells, vascular cells #### Tips and Tricks - Myelin removal is generally necessary : Spinal cord has a higher myelin-to-cell ratio than brain. Without myelin depletion, downstream single-cell workflows commonly fail due to clogging and debris. - Regional segmentation : Cervical, thoracic, and lumbar segments have different cell compositions. Process segments separately if regional specificity is needed. - Thy1 immunopanning : For motor neuron enrichment, Thy1 immunopanning after dissociation significantly improves neuronal proportion (as described in Sloan et al., 2018). - RNA quality : Spinal cord tissue degrades rapidly. Process within 1 hour of collection or snap-freeze immediately. #### Troubleshooting Problem Likely Cause Solution Excessive myelin debris Incomplete myelin removal Double the Percoll gradient or use MACS beads in addition Low neuronal yield Over-digestion Reduce papain to 20 U/mL or shorten to 30 min Cell clumping Insufficient DNase Increase DNase I to 300 U/mL Poor viability after myelin removal Harsh gradient conditions Use gentler 22% single-layer Percoll instead of two-layer #### Singulator Protocol The Singulator's standard nuclei isolation protocol with the Nuclei Isolation Cartridge (blue) is the recommended starting point for spinal cord, as nuclei isolation avoids the myelin removal challenge entirely. For cell isolation, papain can be loaded via the Single-Shot mechanism using the Cell Isolation Cartridge (white), but a post-processing myelin removal step will still be necessary. #### References [1] Literature-standard spinal cord dissociation protocol based on papain digestion with myelin removal (Trevino et al., 2021; Sloan et al., 2018). ### Spleen #### Overview The spleen is the largest secondary lymphoid organ, composed of red pulp (for filtering blood, removing aged RBCs, and storing platelets) and white pulp (organized lymphoid tissue with T cell zones and B cell follicles). The spleen has a relatively soft, friable texture that makes it one of the easiest solid organs to dissociate. The main challenges are the massive number of RBCs (requiring thorough lysis) and the high RNase content (requiring inhibitor for RNA-based applications). #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase D 1 mg/mL Roche 11088858001 Gentle; preserves immune cell surface markers Collagenase IV 0.5 mg/mL Worthington LS004188 Alternative DNase I 50-100 U/mL Roche Prevents clumping RPMI 1640 1x Gibco Standard immune cell medium FBS 2-10% Various Cell protection ACK Lysis Buffer 1x Various CRITICAL -- spleen is full of RBCs RNase Inhibitor 0.4-1 U/uL Sigma Protector Spleen has high RNase activity #### Step-by-Step Protocol Standard Enzymatic Protocol : - Tissue preparation (5 min): Place spleen on ice in cold RPMI. Remove fat and connective tissue. For mouse spleen, the entire organ (60-100 mg) is typically used. - Mechanical pre-dissociation (5 min): Place spleen between frosted glass slides and gently press/smear to release splenocytes. Alternatively, mince with scissors into small fragments and press through a 70 um cell strainer with a syringe plunger. - Optional enzymatic digestion (15-20 min): For stromal cell recovery, incubate fragments in Collagenase D (1 mg/mL) + DNase I (50 U/mL) in RPMI at 37 degrees C. This improves recovery of DCs, stromal cells, and macrophages. - Filtration (5 min): Filter through 70 um cell strainer. Wash with RPMI + 2% FBS. - RBC lysis (5 min): This is the most critical step. Resuspend in 5-10 mL ACK buffer for 5 min at RT. Quench with 15 mL RPMI + 10% FBS. May need to repeat if pellet remains visibly red. - Counting (5 min): Count with AO/PI. Typical mouse spleen yields 50-100 million cells. #### Expected Results - Cell yield : 50-200 million cells per mouse spleen; highly variable for human - Viability : 90-98% (splenocytes are robust) - Common cell types recovered : B cells (dominant, ~50-60%), T cells (CD4+ and CD8+, ~25-30%), dendritic cells, macrophages, NK cells, neutrophils, erythroid precursors, stromal cells (with enzymatic digestion) - Processing time : 20-30 minutes total; ~25 min on Singulator + 15 min post-processing #### Tips and Tricks - Mechanical dissociation alone is often sufficient : Spleen is soft enough that enzymatic digestion is only needed for stromal and DC recovery. For a standard lymphocyte prep, just press through a strainer. - RBC lysis is the bottleneck : Spleen contains more RBCs per gram than almost any other tissue. Plan for 2 rounds of ACK lysis if needed. - RNase inhibitor required for scRNA-seq : Spleen has high RNase expression. 10x Genomics and Precision Cell Systems both recommend RNase inhibitor. - Collagenase D preserves surface markers : Unlike Collagenase I/II, Collagenase D has minimal tryptic activity and preserves CD markers needed for FACS sorting and immunophenotyping. #### Troubleshooting Problem Possible Cause Solution Heavy RBC contamination Insufficient lysis Repeat ACK lysis; extend to 5 min; use fresh ACK buffer Low DC yield Mechanical-only protocol Add Collagenase D enzymatic step (15-20 min at 37 degrees C) Poor RNA quality RNase degradation Add RNase inhibitor to all buffers; process quickly Cell clumping DNA from lysed RBCs Add DNase I to all buffers #### Singulator Protocol Singulator 100/200 -- Mouse Spleen Cell Isolation protocol Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Mouse Spleen Cell Reagent (weaker collagenase formulation) Auto-Mince : Yes Protocol type : Standard Cell Isolation (1 incubation + 1 grind cycle) Incubation : ~25 minutes on instrument Post-processing : Centrifuge 300g x 5 min, ACK RBC lysis (may need 2 rounds), count with AO/PI Notes : Spleen is one of the faster tissue types to process on the Singulator. Plan for thorough RBC lysis post-processing Source : Precision Cell Systems Cell Isolation Protocol Guide; Precision Cell Systems #### References [1] protocols.io: Adult Mouse Spleen Dissociation (On ice) v3. Andrew Potter (CCHMC). https://www.protocols.io/view/adult-mouse-spleen-dissociation-on-ice-ydwfs7e ### Stomach #### Overview Gastric tissue contains a complex epithelium with multiple specialized cell types including parietal cells (acid-secreting), chief cells (pepsinogen-secreting), mucus-producing cells, and stem cells in the gastric glands. The thick mucus layer and acid-resistant epithelium present specific challenges. Applications include gastric cancer research, H. pylori infection studies, and gastric organoid generation. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Collagenase I 2 mg/mL Worthington ECM degradation Dispase II 2.4 U/mL Roche Epithelial release DNase I 100 U/mL Roche Prevent clumping DTT 10 mM -- Mucus removal EDTA 10 mM -- Epithelial loosening HBSS without Ca/Mg -- Gibco EDTA chelation buffer HBSS with Ca/Mg -- Gibco Enzyme digestion buffer N-acetylcysteine 10 mM Sigma Mucus dissolution #### Step-by-Step Protocol - Mucus removal (20 min): Incubate tissue in DTT (10 mM) or N-acetylcysteine (10 mM) in HBSS at room temperature to dissolve the mucus layer. - Epithelial stripping (30 min): Transfer to EDTA (10 mM) in HBSS without Ca/Mg at 37 degrees C with gentle rocking. Collect supernatant fractions every 10 minutes (contains epithelial cells). - Stromal digestion (30 min): Digest remaining tissue in Collagenase I (2 mg/mL) + Dispase II (2.4 U/mL) + DNase I (100 U/mL) in HBSS with Ca/Mg at 37 degrees C. - Combine fractions (5 min): Pool epithelial (EDTA) and stromal (collagenase) fractions if full cellular representation is needed. - Filtration (5 min): Filter through 70 um strainer. - Wash and count (10 min): Centrifuge 300g x 5 min, resuspend in PBS + 0.04% BSA, count. #### Expected Results - Cell yield : 1-5 x 10^6 cells per biopsy or per 100 mg tissue - Viability : 75-90% - Common cell types recovered : Parietal cells, chief cells, mucous neck cells, surface mucous cells, gastric stem/progenitor cells (Lgr5+), enteroendocrine cells, immune cells, fibroblasts, smooth muscle cells #### Tips and Tricks - Mucus removal is important : Without DTT or N-acetylcysteine pre-treatment, thick gastric mucus can trap cells and create aggregates. - Two-fraction approach : The EDTA fraction enriches for epithelial cells while the collagenase fraction captures stromal and immune populations. Keep them separate if specific populations are targeted. - Acid damage : Gastric tissue from the corpus may have low-pH pockets. Rinse thoroughly in neutral buffer before digestion. - Tumor tissue : Gastric cancer tissue is typically firmer and may require extended collagenase digestion (45-60 min). #### Troubleshooting Problem Likely Cause Solution Mucus aggregates Insufficient DTT treatment Extend DTT to 30 min or increase to 15 mM Low epithelial yield EDTA too brief Collect more EDTA fractions over longer period Dead parietal cells Acid exposure during prep Rinse tissue 3x in neutral HBSS immediately Excessive debris Connective tissue fragments Add 40 um filtration step Stomach cancer tissue has been processed using the Singulator's standard nuclei isolation protocol. Human stomach cancer tissue (~30 mg) yielded nuclei stained with DAPI and visualized by confocal microscopy. For nuclei isolation, the standard protocol with the Nuclei Isolation Cartridge (blue) applies directly. For cell isolation, the two-step mucus removal + enzymatic digestion approach can be adapted, with the collagenase/Dispase cocktail loaded via the Single-Shot mechanism (3 mL enzyme + 6 mL buffer) using the Cell Isolation Cartridge (white). #### References [1] Literature-standard gastric tissue dissociation protocol based on EDTA chelation + collagenase digestion approach (Barker et al., 2010; Stange et al., 2013). ### Tendon / Ligament #### Overview Tendon and ligament tissues are among the most challenging to dissociate due to their dense, highly organized collagen fiber architecture and relatively low cellularity. Tenocytes reside between tightly packed collagen fibers and are difficult to liberate without compromising viability. Applications include tendon injury repair research, tendinopathy studies, and tissue engineering. Extended enzymatic digestion (2+ hours) is typically required. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Collagenase I 3 mg/mL Worthington Primary collagen degradation Collagenase II 3 mg/mL Worthington Complementary collagen types Dispase II 4 mg/mL Roche Cell liberation DNase I 100 U/mL Roche Prevent clumping Liberase TH 1 mg/mL Roche Alternative one-enzyme approach DMEM/F12 -- Gibco Digestion medium FBS 10% -- Enzyme quenching #### Step-by-Step Protocol - Tissue preparation (15 min): Remove paratenon/adventitia. Mince tendon or ligament into the smallest possible pieces ( #### Expected Results - Cell yield : 50,000-500,000 cells per tendon (highly variable by tissue mass and digestion completeness) - Viability : 60-80% (lower than most tissues due to extended digestion) - Common cell types recovered : Tenocytes, tendon stem/progenitor cells, endothelial cells, pericytes, immune cells (macrophages), fibroblasts #### Tips and Tricks - Thorough mincing is the rate-limiting step : The denser the collagen packing, the more critical fine mincing becomes. Use crossed scalpels for 5+ minutes. - Tissue mass matters : Testing showed that 50 mg tendon in 5 mL enzyme solution led to complete cell death, while 210 mg in 5 mL reached complete digestion in 2 hours but with high debris. - Optimal mass range : 70-100 mg of tendon tissue in 5 mL enzyme solution provides the best balance of yield and viability. - Debris removal : Tendon digests generate significant debris. A debris removal solution step may be necessary after filtration. - Rat vs. human : Rat tendons digest more readily (60-90 min) than human tendons (2+ hours). #### Troubleshooting Problem Likely Cause Solution Incomplete digestion Insufficient mincing or enzyme Mince more finely; increase collagenase to 4 mg/mL Complete cell death Too much enzyme per tissue mass Increase tissue-to-enzyme volume ratio (100+ mg per 5 mL) Excessive debris Complete digestion of collagen Use debris removal solution or Percoll gradient Low viability Extended digestion time Try Liberase TH (1 mg/mL) for faster, gentler digestion Clumping DNA from dead cells Increase DNase I to 200 U/mL #### Singulator Protocol A Cedars-Sinai research team (Ahmet Pazarceviren and Chintda Santiskulvong) extensively tested tendon tissue dissociation on the Singulator. Key findings: (1) The Singulator achieved complete digestion of tendon tissue without the need for debris removal solution, demonstrating excellent mechanical disruption. (2) Liberase + DNase concentration was sufficient for 70 mg tendon. (3) A 30-minute digestion was proposed to improve viability over longer protocols. (4) The researcher used the Mouse Lung Cell Isolation protocol as a base (2 incubation + 2 grind cycles) to adapt for tendon. The Cell Isolation Cartridge (white) with Liberase TH (1 mg/mL) loaded via Single-Shot (3 mL enzyme + 6 mL buffer) is recommended. #### References [1] Literature-standard tendon dissociation protocol based on high-concentration collagenase digestion approach (Bi et al., 2007; Tan et al., 2012). ### Testis #### Overview The testis contains seminiferous tubules (with spermatogonia, spermatocytes, spermatids, and Sertoli cells) surrounded by interstitial tissue (Leydig cells, macrophages, blood vessels, fibroblasts). Dissociation requires penetration of the tunica albuginea (dense collagenous capsule) and the basement membrane of seminiferous tubules. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 0.5-1 mg/mL Worthington LS004188 For interstitial digestion Trypsin-EDTA 0.25% Gibco For tubule dissociation DNase I 50-100 U/mL Roche Anti-clumping (critical -- many dead spermatids) HBSS 1x Various Base buffer #### Step-by-Step Protocol - Tissue preparation (10 min): Remove tunica albuginea. Tease apart seminiferous tubules with fine forceps. - First digest (15-20 min): Collagenase IV (0.5 mg/mL) in HBSS at 37 degrees C to separate tubules from interstitium. - Second digest (15-20 min): Transfer tubules to Trypsin-EDTA (0.25%) + DNase I (100 U/mL) at 37 degrees C to dissociate tubular cells. - Quench and filter (5 min): Add FBS. Filter through 70 um then 40 um strainers. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 5-50 million cells per mouse testis - Viability : 80-95% - Common cell types recovered : Spermatogonia, spermatocytes, round spermatids, elongating spermatids, Sertoli cells, Leydig cells, peritubular myoid cells, macrophages, endothelial cells #### Singulator Protocol Singulator 100/200 -- Custom two-step protocol using open reagent platform. #### References [1] protocols.io: Adult mouse testis cell dissociation (on ice). CCHMC. https://www.protocols.io/view/adult-mouse-testis-cell-dissociation-on-ice-smkec4w ### Thymus #### Overview The thymus is a primary lymphoid organ where T cell maturation occurs, organized into a cortex (dense immature thymocytes) and medulla (mature thymocytes, medullary thymic epithelial cells, dendritic cells, macrophages). Like spleen and lymph node, the thymus is relatively soft and yields large numbers of cells with minimal processing. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase D 1 mg/mL Roche 11088858001 For epithelial cell recovery Liberase TH 0.1 mg/mL Roche Gentle alternative DNase I 50 U/mL Roche Anti-clumping RPMI 1x Various Base medium #### Step-by-Step Protocol - Mechanical dissociation (5 min): Press thymus through 70 um cell strainer with syringe plunger. This releases >90% of thymocytes. - Optional enzymatic digestion (20-30 min): For thymic epithelial cells (TECs), mince remaining tissue and digest with Collagenase D (1 mg/mL) + DNase I (50 U/mL) at 37 degrees C. - Filtration (5 min): Filter through 40 um strainer. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 50-200 million cells per mouse thymus (mostly thymocytes) - Viability : >95% - Common cell types recovered : CD4+CD8+ double-positive thymocytes (majority), CD4SP, CD8SP, double-negative thymocytes, cortical TECs (cTECs), medullary TECs (mTECs), dendritic cells, macrophages, B cells #### Singulator Protocol The thymus, like spleen and lymph node, is soft enough that mechanical dissociation through a strainer is typically sufficient. The Singulator is not usually needed but can be used with gentle settings for standardized processing. #### References [1] protocols.io: Human Thymus single cell dissociation protocol. Teichmann Lab (Sanger). https://www.protocols.io/view/human-thymus-single-cell-dissociation-protocol-tei-bx8sprwe ### Thyroid #### Overview The thyroid gland consists of follicles lined by follicular epithelial cells (thyrocytes) surrounding colloid, plus parafollicular C cells, fibroblasts, endothelial cells, and immune cells. The follicular architecture requires collagenase-based digestion. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase II 1 mg/mL Worthington LS004176 Standard for thyroid Dispase II 1 U/mL Roche For follicular dissociation DNase I 50-100 U/mL Roche Anti-clumping #### Step-by-Step Protocol - Tissue preparation (10 min): Remove capsule and connective tissue. Mince into 1-2 mm pieces. - Enzymatic digestion (45-60 min): Collagenase II (1 mg/mL) + Dispase II (1 U/mL) + DNase I (50 U/mL) at 37 degrees C with agitation. - Filtration (5 min): Filter through 70 um strainer. - Counting (5 min): Count with AO/PI. #### Expected Results - Cell yield : 1-10 million cells per gram - Viability : 80-90% - Common cell types recovered : Follicular epithelial cells (thyrocytes), C cells, fibroblasts, endothelial cells, immune cells #### Singulator Protocol Singulator 100/200 -- Custom protocol via open reagent platform with collagenase/dispase cocktail. #### References [1] Landa I, et al. (2016). Genomic and transcriptomic hallmarks of poorly differentiated and anaplastic thyroid cancers. Journal of Clinical Investigation 126(3): 1052-1066. DOI: 10.1172/JCI85271. ### Trachea / Airway #### Overview Tracheal and airway tissue features a pseudostratified columnar epithelium rich in ciliated cells, goblet cells, and basal stem cells, underlaid by a submucosa with cartilaginous rings. Dissociation typically targets either the airway epithelium (via pronase or EDTA stripping) or the full-thickness tissue (via collagenase). Basal cells are of particular interest for airway regeneration research and air-liquid interface (ALI) culture. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Pronase 1.5 mg/mL Roche Epithelial stripping Collagenase IV 2 mg/mL Worthington Full-thickness digestion DNase I 100 U/mL Roche Prevent clumping EDTA 5 mM -- Epithelial loosening FBS 10% -- Pronase neutralization DMEM/F12 -- Gibco Digestion medium BEGM -- Lonza Basal cell culture medium #### Step-by-Step Protocol (Epithelial Isolation) - Tissue preparation (10 min): Open trachea or bronchus longitudinally. Rinse luminal surface with PBS to remove mucus. - Pronase digestion (overnight or 2 hours): For overnight, incubate tissue in Pronase (1.5 mg/mL) in DMEM/F12 at 4 degrees C for 16-18 hours. For a rapid protocol, use Pronase at 37 degrees C for 2 hours. - Cell harvest (10 min): Add FBS to 10% to neutralize pronase. Gently scrape epithelial surface with cell scraper. Collect cells by pipetting. - Wash (5 min): Centrifuge 300g x 5 min, resuspend in BEGM or PBS. - DNase treatment (5 min): If clumps persist, treat with DNase I (100 U/mL) for 5 minutes. - Filter and count (5 min): Filter through 40 um strainer, count. #### Expected Results - Cell yield : 0.5-2 x 10^6 epithelial cells per cm of mouse trachea; 1-5 x 10^6 per human bronchial segment - Viability : 85-95% - Common cell types recovered : Basal cells (p63+/KRT5+), ciliated cells, goblet/secretory cells, club cells, neuroendocrine cells, ionocytes (rare), submucosal gland cells #### Tips and Tricks - Overnight 4 degrees C pronase is gentlest : This approach gives highest viability for basal cells compared to 37 degrees C digestion. - Cartilage handling : Do not attempt to digest cartilaginous rings. Either strip epithelium from the luminal side or carefully dissect mucosa away from cartilage. - Mucus removal : Thorough PBS rinses of the luminal surface before pronase treatment improve epithelial access. - Basal cell enrichment : After dissociation, plate on collagen-coated dishes for 4 hours -- basal cells adhere preferentially. #### Troubleshooting Problem Likely Cause Solution Low epithelial yield Incomplete pronase exposure Ensure full luminal surface contact; extend to 20 hours at 4C Mucus contamination Insufficient pre-rinse Rinse 5+ times with warm PBS before pronase Cartilage fragments Over-aggressive processing Strip epithelium only; do not mince through cartilage Poor basal cell growth Dead differentiated cells Pre-plate to remove debris; use BEGM with Y-27632 ROCK inhibitor #### Singulator Protocol Tracheal and airway tissue have limited direct mentions in the Precision Cell Systems PM archive. However, the Singulator's lung cell isolation protocol architecture (2 incubation + 2 grind cycles) could serve as a starting point for full-thickness airway tissue using Collagenase IV loaded via Single-Shot. For epithelial-only isolation, the overnight pronase approach cannot be replicated on the instrument, but the post-pronase mechanical harvesting and filtration steps could benefit from the Singulator's standardized processing with the Cell Isolation Cartridge (white). #### References [1] Literature-standard airway epithelial dissociation protocol based on pronase stripping approach (You et al., 2002; Rock et al., 2009; Plasschaert et al., 2018). # Priority 4: Model Organisms and Engineered Tissues ### Tumor (General Solid Tumors) #### Overview Solid tumors are among the most challenging tissues to dissociate due to their extreme heterogeneity in ECM composition, cellularity, necrosis, fibrosis (desmoplastic stroma), and vascularization. Tumors contain cancer cells, cancer-associated fibroblasts (CAFs), tumor-associated macrophages (TAMs), T cells (including TILs), B cells, NK cells, dendritic cells, endothelial cells, pericytes, and smooth muscle cells. The dissociation protocol must handle dense stroma while preserving fragile immune cell populations and maintaining cell surface markers for phenotyping. #### Recommended Enzymes/Reagents Enzyme/Reagent Concentration Source/Catalog # Notes Collagenase IV 1-2 mg/mL Worthington LS004188 Broad-spectrum; preserves surface markers Collagenase I 2 mg/mL Sigma C0130 For dense fibrous tumors Hyaluronidase 100-300 U/mL Sigma H3506 Breaks down tumor stroma DNase I 100-200 U/mL Roche High concentration needed due to necrosis Liberase TH 1 mg/mL Roche Alternative; validated for many tumor types RPMI or DMEM 1x Various Base medium FBS 5-10% Various Cell protection ACK Lysis Buffer 1x Various For vascularized tumors Percoll 25-50% Various Dead cell and debris removal #### Step-by-Step Protocol - Tissue preparation (15 min): Place tumor on ice immediately. Document size, appearance, consistency. Remove necrotic regions (soft, discolored areas). Mince viable regions into 1-2 mm pieces with crossed scalpels. - Enzymatic digestion (30-60 min): Incubate in Collagenase IV (1 mg/mL) + Hyaluronidase (200 U/mL) + DNase I (100 U/mL) in RPMI + 5% FBS at 37 degrees C with rotation (180 rpm). Duration depends on tumor density -- check every 15 min. - Trituration (5 min): Triturate with 10 mL serological pipette, then 1000 uL pipette tip (cut if needed). Avoid excessive force. - Filtration (5 min): Filter through 100 um then 70 um strainers. Wash with cold RPMI. - RBC lysis (5 min): ACK buffer 3-5 min at RT if tumor is vascularized. - Dead cell/debris removal (15 min): Percoll density gradient centrifugation (300g, 10 min, brake off) to remove debris and necrotic cells. - Counting (5 min): Count with AO/PI. Note viability -- target >70% for scRNA-seq. #### Expected Results - Cell yield : 5,000,000-6,700,000 cells per sample (human lung tumor, Singulator); highly variable by tumor type - Viability : 76-99% (Singulator platform across 3 lung tumor patients); 60-80% is typical for manual protocols - Common cell types recovered : Cancer cells (variable), CAFs, TAMs, T cells (CD4+, CD8+, Tregs), B cells, NK cells, DCs, endothelial cells, pericytes - Processing time : 45-90 minutes total; uses Mouse Lung Cell Isolation protocol on Singulator #### Tips and Tricks - Tumor heterogeneity demands multi-region sampling : Take samples from tumor periphery, core, and invasion front for representative profiling. - Necrosis removal is important : Necrotic tissue commonly contributes dead cells, DNA debris, and ambient RNA. Thorough trimming before mincing is recommended. - Pan Tumor Reagent is the most aggressive Precision Cell Systems formulation : Designed for dense tumor stroma. - TIL surface markers preserved : The Singulator platform preserves CD45 epitopes, enabling downstream TIL enrichment via immunomagnetic selection (e.g., StemCell EasySep CD45+). - Tumor type matters : Soft tumors (brain, lymphoma) require less aggressive treatment; dense fibrotic tumors (pancreatic, breast desmoplastic) need maximal enzyme and time. - For the Singulator, use the Mouse Lung Cell Isolation protocol with Pan Tumor Reagent : This dual-incubation protocol provides the aggressive processing needed for most solid tumors. #### Troubleshooting Problem Possible Cause Solution Dense tumor resistant to digestion Desmoplastic stroma Increase Collagenase to 2 mg/mL; add Hyaluronidase; extend time to 60 min Low viability Necrotic tissue included Remove necrotic regions more aggressively; use dead cell removal Immune cell loss Over-digestion damaging lymphocytes Reduce digestion time; use sequential collection (remove supernatant at 30 min, add fresh enzyme) High debris in scRNA-seq Insufficient cleanup Add Percoll gradient step; filter through 40 um strainer Cell surface marker loss Prolonged enzymatic exposure Reduce digestion time; use Collagenase IV (lower tryptic activity) #### Singulator Protocol Singulator 100/200 -- Mouse Lung Cell Isolation protocol with Pan Tumor Reagent Cartridge : Cell Isolation Cartridge (100-063-178, white top) Reagent : Pan Tumor Reagent (100-247-099) Auto-Mince : Yes Sample : Fresh tumor tissue, finely minced, washed with cold PBS Protocol type : Mouse Lung Cell Isolation (2 incubation + 2 grind cycles) Incubation : 37 degrees C, dual incubation cycle Post-processing : Centrifuge 300g x 5 min, ACK RBC lysis, Percoll density gradient debris removal, sequential filtration through cell strainers, count with AO/PI Results : From 3 human lung tumor patients -- 5,880,000, 6,732,000, and 5,065,000 cells at 97%, 94%, and 76% viability respectively. scRNA-seq: 6,458-8,500 cells per library with cell type representation matching tumor pathology Source : Precision Cell Systems Application Note: Dissociating Tumor Tissue into Cells with Precision Cell Systems' Pan Tumor Reagent (P/N: 100-261-619) #### References [1] protocols.io: Single Cell Dissociation of Small Tumor Biopsies. ETH Zurich/CZ Biohub. https://www.protocols.io/view/single-cell-dissociation-of-small-tumor-biopsies-65rhg56 ### Zebrafish #### Overview Zebrafish are widely used model organisms for developmental biology, regeneration, and disease modeling. Dissociation of embryos, larvae, and adult tissues requires protocols adapted to the developmental stage. Embryos and early larvae can be dissociated relatively easily, while adult tissues with scales and calcified structures require more aggressive treatment. A key consideration is the optimal incubation temperature of 28-32 degrees C (zebrafish physiological range) rather than the 37 degrees C used for mammalian tissues. #### Recommended Enzymes and Reagents Reagent Concentration Source Purpose Trypsin-EDTA 0.25% Gibco Embryo/larval dissociation Collagenase P 1 mg/mL Roche Adult tissue dissociation Liberase TM 0.5 mg/mL Roche Gentle alternative DNase I 100 U/mL Roche Prevent clumping PBS without Ca/Mg -- -- Trypsin digestion buffer Tricaine (MS-222) 0.02% Sigma Anesthesia Pronase 2 mg/mL Roche Chorion removal (embryos) FBS 10% -- Trypsin neutralization Calcium-free Ringer's -- -- Epithelial loosening #### Step-by-Step Protocol (Embryos/Larvae) - Dechorionation (embryos only, 5 min): Treat embryos with Pronase (2 mg/mL) in E3 medium until chorions soften, then pipette gently to remove. - Anesthesia and collection (5 min): Anesthetize larvae in Tricaine (0.02%), collect in a microcentrifuge tube, remove excess medium. - Enzymatic digestion (25-30 min): Add 0.25% Trypsin-EDTA. Incubate at 28-32 degrees C. Manually disrupt tissue every 5 minutes by pipetting. - Trypsin neutralization (2 min): Add equal volume of PBS + 10% FBS or soybean trypsin inhibitor. - Filtration (5 min): Filter through 40 um strainer to remove debris and undigested fragments. - Wash and count (10 min): Centrifuge 300g x 5 min, resuspend in PBS + 0.04% BSA, count. #### Expected Results - Cell yield : 50,000-200,000 cells per embryo (stage-dependent); 100,000-500,000 per larva (8-12 dpf) - Viability : 80-95% for embryos; 70-85% for larvae - Common cell types recovered : Variable by stage -- skin cells, blood cells, muscle progenitors, neurons, notochord cells, endoderm derivatives, immune cells #### Tips and Tricks - Temperature matters : Zebrafish cells are adapted to 28 degrees C. Incubation at 37 degrees C can damage cells. A researcher specifically asked about using room temperature on the Singulator rather than heating to 37 degrees C. - Developmental stage : Younger embryos ( #### Troubleshooting Problem Likely Cause Solution Incomplete dissociation of larvae Insufficient trypsin exposure Extend to 35 min; increase trituration frequency Low viability Temperature too high Reduce to 28 degrees C; confirm incubator setting Melanocyte interference Pigment granule autofluorescence Pre-treat with PTU or use non-fluorescent sorting parameters Yolk granule contamination Early embryos ( Allow yolk to settle by gravity, collect supernatant Skeletal debris in adults Calcified structures Pre-filter through 100 um before 40 um #### Singulator Protocol A researcher at the Stowers Institute (a researcher) specifically inquired about dissociating zebrafish embryos (8-12 days old) on the Singulator. Key discussion points: (1) The mouse lung cell isolation protocol (2 incubation + 2 grind cycles) was suggested as a base, with a 12-15 minute incubation period; (2) Temperature optimization was discussed -- room temperature rather than 37 degrees C was proposed to match zebrafish physiology; (3) Mixing type modifications were considered given the researcher's manual protocol disrupted tissue every 5 minutes. Additional zebrafish interest has come from labs at the University of Oregon (enzymatic digestion and caudal fin preparations) and the Guo lab for zebrafish single nuclei extraction. The Cell Isolation Cartridge (white) with 0.25% Trypsin-EDTA loaded via Single-Shot would be the starting configuration. #### References [1] protocols.io: Zebrafish Embryo Dissociation for MACS v2. Harvard/BCH. https://www.protocols.io/view/zebrafish-embryo-dissociation-for-macs-bhquj5ww [2] protocols.io: Zebrafish larvae dissociation for FACS sorting. Edinburgh. https://www.protocols.io/view/zebrafish-larvae-dissociation-for-facs-sorting-cel-bzh8p39w Nuclei Isolation ## Tissue-to-Nuclei Protocols 42 tissue types with lysis buffers, Dounce homogenization, and filtration protocols for single-nucleus analysis. Select Tissue Type Adipose Adrenal Aorta / Vasculature Bladder Bone Bone Marrow Brain C. elegans Cartilage Cochlea Colon / Intestine Cornea Dorsal Root Ganglia Drosophila Embryonic Tissue Endometrium Esophagus FFPE Tissue Heart iPSC-Derived Tissues Kidney Liver Lung Lymph Node Muscle (Skeletal) Organoids Ovary Pancreas Peripheral Nerve Placenta Prostate Retina Salivary Gland Skin Spinal Cord Spleen Stomach Testis Thymus Thyroid Tumors (Solid) Zebrafish ### Adipose #### Overview Adipose tissue is composed predominantly of large, lipid-filled adipocytes (50--100 um diameter) embedded in a loose connective tissue matrix containing the stromal vascular fraction (SVF) -- preadipocytes, endothelial cells, immune cells (macrophages, T cells), and fibroblasts. The high lipid content creates two major challenges for nuclei isolation: (1) released lipids form a floating debris layer that interferes with pelleting, and (2) adipocyte nuclei are more fragile than nuclei from other cell types, likely due to the mechanical stress of lipid release. Nuclei isolation from adipose has been validated on the Singulator, though yields are lower than denser tissues. The Standard Nuclei Isolation protocol is the recommended starting point, with modifications for higher centrifugation speed and careful lipid debris removal. #### Recommended Lysis Buffer Standard NP-40 lysis buffer with VRC supplementation (recommended per recent adipose snRNA-seq protocols): Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein, also helps block lipid binding DTT 1 mM Reducing agent RNase inhibitor 0.2--1.0 U/uL 0.2 U/uL satisfactory; 1.0 U/uL for stronger protection VRC (optional) 10 mM Additional RNase inhibition recommended for adipose #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen adipose tissue. Weigh 50--300 mg (adipose is low-density, so more tissue mass is needed). - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. Adipose tends to float; ensure tissue contacts the buffer. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. Adipose disrupts readily. - Washing : Centrifuge 700--1,000g, 5 min, 4 degrees C (higher speed than standard 500g because adipocyte nuclei pellet poorly). - Lipid removal : Carefully remove floating lipid layer with wide bore pipette before aspirating supernatant. Use Kimwipe-wrapped serological pipette to soak up lipid layer if needed. - Repeat wash : Resuspend in wash buffer. Centrifuge again at 700--1,000g. Remove lipid layer. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining and counting. Count in NSR + 1% BSA. #### Expected Results - Nuclei yield : Low; 1,000--7,000 nuclei per mg from literature; yields from adipose are consistently lower than denser tissues - Cell type composition : Adipocyte nuclei, preadipocytes, macrophages, endothelial cells, T cells, fibroblasts - Processing time : ~7 min Singulator plus downstream processing - Downstream compatibility : 10x Chromium (can load in NSR + 1% BSA to avoid buffer exchanges) #### Tips and Tricks - Higher centrifugation speed essential : Standard 500g is often insufficient to pellet adipocyte nuclei. Use 700--1,000g for 5 min. - Swinging bucket centrifuge with brake OFF : Use swinging bucket rotor for density-based debris removal to prevent mixing of lipid layer. - Modified NIR delivery : Mince tissue, add approximately 500 uL of buffer, then run Low Volume protocol. Alternatively, modify NIR delivery volume to 1.5 mL in Standard protocol. - NSR + 1% BSA for downstream : Nuclei can be loaded onto 10x in NSR + 1% BSA, avoiding unnecessary buffer exchanges that risk losing the already low-yield sample. - Extended protocol if yield is low : Try Extended Nuclei Isolation if Standard yields are insufficient. - Flow cytometry-assisted cleanup : A 2024 protocol integrates FACS for quality control, nuclei counting, and precise pooling, which eliminates ambient RNA contamination and poor-quality nuclei from adipose preparations. #### Troubleshooting Problem Possible Cause Solution Nuclei fail to pellet Lipid interference Increase centrifugation to 700--1,000g; use swinging bucket rotor Floating lipid debris Released adipocyte lipid Remove lipid layer with wide bore pipette or Kimwipe-wrapped serological pipette Very low yield Inherent to adipose tissue Increase starting material (200+ mg); try Extended protocol Nuclei damage Over-disruption Reduce Singulator speed to medium or slow #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard or Extended Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.2--1.0 U/uL) Sample size : 50--300 mg frozen adipose tissue Auto-Mince : Yes Incubation : 0 min (Standard) or 5 min at Cold (Extended for low yield) Mixing : Top at Fastest speed (reduce if nuclei appear damaged) Disruption : Default at Fastest speed (reduce to Medium if nuclei appear damaged) Post-processing : Centrifuge 700--1,000g, 5 min, 4 degrees C; remove floating lipid with wide bore pipette; resuspend in NSR + 1% BSA + RNase inhibitor; filter through 40 um strainer Run time : ~7 min (Standard) or ~12 min (Extended) on instrument Modification option : Use Low Volume protocol with 500 uL of buffer pre-added to minced tissue #### References [1] Benitez GJ, Shinoda K. Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications. Journal of Visualized Experiments. 2020;(160):e61230. [https://pmc.ncbi.nlm.nih.gov/articles/PMC7971773/](https://pmc.ncbi.nlm.nih.gov/articles/PMC7971773/) [2] Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) ### Adrenal #### Overview The adrenal gland has two distinct regions: the cortex (zona glomerulosa, zona fasciculata, zona reticularis -- producing mineralocorticoids, glucocorticoids, and androgens) and the medulla (chromaffin cells producing catecholamines). The tissue is soft and moderately cellular. Standard nuclei isolation protocols work well. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 10--15 per pestle - Singulator protocol : Standard Nuclei Isolation V2 - Cartridge : NIC+ (adrenal glands are typically small, 5--20 mg in mice) - Expected yield : Moderate (20,000--80,000 nuclei/mg) - Key cell types : Cortical steroidogenic cells, chromaffin cells, endothelial cells, immune cells ### Aorta / Vasculature #### Overview Vascular tissue consists of three layers: intima (endothelial cells), media (smooth muscle cells, elastic laminae), and adventitia (fibroblasts, vasa vasorum, nerves). The high elastin and collagen content makes vascular tissue mechanically resistant. Extended Nuclei Isolation with pre-mincing is recommended. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1--0.2%) - Dounce strokes : 15--20 per pestle - Singulator protocol : Extended Nuclei Isolation V2 (5 min incubation, dual disruption) - Cartridge : NIC+ for small vessel segments - Expected yield : Low to moderate (5,000--30,000 nuclei/mg due to low cellularity) - Key cell types : Endothelial cells, smooth muscle cells, fibroblasts, macrophages, pericytes - Tips : Mince very finely due to elastic resistance; Extended protocol recommended ### Bladder #### Overview Bladder tissue has a layered architecture: urothelium (transitional epithelium, 3--7 cell layers), lamina propria, and detrusor muscle (smooth muscle). The urothelium is moderately resistant to lysis. A 2023 publication optimized snRNA-seq from frozen human bladder tumor biopsies using IGEPAL lysis buffer with tissue sectioning and short incubation. #### Recommended Protocol - Lysis buffer : IGEPAL CA-630 (0.1%), 10 mM Tris-HCl, 10 mM NaCl, 3 mM MgCl2 - Dounce strokes : 10--15 per pestle - Singulator protocol : Standard or Extended Nuclei Isolation V2 - Cartridge : NIC+ for biopsies - Expected yield : Moderate (20,000--60,000 nuclei/mg) - Key cell types : Urothelial cells, smooth muscle cells, fibroblasts, immune cells (T cells, macrophages) - Tips : Short lysis incubation (5 min); sectioning technique improves nuclei quality for biopsies #### References [1] Haegebarth A, et al. Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies. Nucleus. 2023;14(1):2186686. [https://pmc.ncbi.nlm.nih.gov/articles/PMC10012951/](https://pmc.ncbi.nlm.nih.gov/articles/PMC10012951/) ### Bone #### Overview Bone tissue presents extreme challenges for nuclei isolation. The mineralized matrix requires either physical pulverization (mortar and pestle with liquid nitrogen) or demineralization before nuclei can be released. The osteocytes embedded in lacunae are difficult to access. For trabecular bone, the marrow can be flushed out first, and the remaining bone matrix pulverized. #### Recommended Protocol - Sample preparation : Pulverize frozen bone in liquid nitrogen using mortar and pestle until powdered - Lysis buffer : Standard NP-40 (0.1%) applied to bone powder - Dounce strokes : 10--15 per pestle (on pulverized powder) - Singulator protocol : Extended Nuclei Isolation V2 on pulverized bone powder - Cartridge : NIC+ - Expected yield : Very low (1,000--10,000 nuclei/mg depending on bone type) - Key cell types : Osteocytes, osteoblasts (surface), osteoclasts, endothelial cells, marrow cells (if not flushed) - Tips : Pre-pulverization in liquid nitrogen is typically necessary; bone matrix generally does not disrupt adequately in Dounce alone ### Bone Marrow #### Overview Bone marrow is a semi-liquid tissue that is easily disrupted mechanically because cells are loosely organized within the marrow cavity. Most bone marrow samples arrive as aspirates (liquid) or as small tissue fragments. Nuclei isolation from bone marrow aspirates or cryopreserved marrow requires minimal mechanical force -- in many cases, simple lysis of the cell suspension is sufficient without Dounce homogenization. The primary cell types recovered include hematopoietic stem cells (HSCs), progenitor cells across all lineages, mature myeloid and lymphoid cells, mesenchymal stromal cells, adipocytes, and erythroid precursors. The Singulator can process bone marrow when the sample is resuspended in NIR and loaded into the cartridge. RBC lysis is strongly recommended as bone marrow is typically blood-rich. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis detergent BSA 1% Carrier protein RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Sample preparation (aspirate) : Thaw cryopreserved bone marrow aspirate on ice. Centrifuge 300g, 5 min, 4 degrees C to pellet cells. Remove supernatant. - Sample preparation (tissue fragment) : On dry ice, mince frozen bone marrow fragments into small pieces. - Lysis : Add 1 mL cold lysis buffer. For aspirates, pipette gently to resuspend. For tissue, use Dounce homogenizer with 5--10 gentle strokes (Pestle A only; minimal force needed). - Incubation : 5 min on ice. - Washing : Centrifuge 500g, 5 min, 4 degrees C. - RBC lysis : Resuspend in ACK lysis buffer, 3 min on ice. Centrifuge 500g, 5 min. - Additional wash : Resuspend in wash buffer. Centrifuge. Repeat. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : High; bone marrow is highly cellular (50,000--500,000 nuclei per mg of tissue fragment; aspirates yield millions of nuclei per mL) - Cell type composition : HSCs, progenitor cells, mature myeloid and lymphoid cells, mesenchymal stromal cells, erythroid precursors - Downstream compatibility : 10x Chromium, BD Rhapsody, CITE-seq #### Tips and Tricks - Minimal mechanical force : Bone marrow cells are loosely organized. Over-disruption damages fragile progenitor cells. - RBC lysis is strongly recommended : Bone marrow is typically heavily contaminated with red blood cells. Multiple ACK lysis cycles may be needed. - Cryopreserved samples work well : Frozen bone marrow aspirates are commonly used for snRNA-seq with good results. - Load as cell suspension : For Singulator processing, resuspend pelleted cells in approximately 100--200 uL NIR and load directly into the dissociation chamber. #### Troubleshooting Problem Possible Cause Solution Heavy RBC contamination Blood-rich tissue Multiple ACK lysis cycles; Ficoll density gradient for aspirates Low viability Freeze-thaw damage to delicate progenitors Process quickly after thawing; keep on ice at all times Clumping DNA release from damaged cells Add DNase I (100 U/mL) to lysis buffer #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample preparation : Resuspend cell pellet or minced tissue in 100--200 uL NIR; load into dissociation chamber Auto-Mince : Yes (for tissue fragments); optional for cell suspensions Incubation : 0 min at Cold Mixing : Top at Slowest speed (bone marrow is delicate) Disruption : Default at Slowest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; ACK lysis for RBC removal; resuspend in NSR + RNase inhibitor; filter through 40 um strainer Run time : ~7 min on instrument ### Brain #### Overview Brain tissue is the most extensively studied tissue for nuclei isolation and snRNA-seq. Nuclei isolation is strongly preferred over cell dissociation for brain because: (1) neurons are extremely large and fragile, making them underrepresented in single-cell suspensions; (2) enzymatic dissociation at 37 degrees C induces immediate early gene expression artifacts; and (3) most brain biobanks store frozen tissue. The primary tissue-specific challenge is myelin debris, which is abundant in white matter and must be removed via density gradient or debris removal reagents. Brain sub-regions (cerebellum, cortex, hippocampus, hypothalamus, cerebral organoids) all use similar base protocols with minor modifications to disruption intensity. #### Recommended Lysis Buffer Standard NP-40 lysis buffer is the most common choice for brain nuclei: Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Primary lysis detergent Tween-20 0.1% Anti-aggregation Digitonin 0.01% Gentle permeabilization BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.2--1.0 U/uL RNA preservation Alternative : Sigma Nuclei EZ Prep kit is widely used for brain and produces consistent results without custom buffer preparation. #### Step-by-Step Protocol (Manual Dounce Method) - Tissue preparation : On dry ice, cut frozen brain tissue into half-rice-grain-sized pieces using a clean, RNase-free blade. Weigh 20--100 mg of tissue. Keep tissue frozen until lysis. - Lysis : Add 600 uL--1 mL cold lysis buffer to a pre-chilled 2 mL Dounce homogenizer. Transfer tissue pieces into buffer on ice. - Mechanical disruption : Homogenize with Pestle A (loose), 10--15 gentle strokes. Switch to Pestle B (tight), 10--15 strokes. Do not create bubbles or remove pestle from liquid. - Incubation (optional): Incubate on ice for 5 min if tissue is insufficiently lysed. Avoid exceeding 15 min total lysis time. - Filtration : Strain through a 35--40 um cell strainer into a pre-chilled tube. Rinse strainer with 500 uL wash buffer. - Washing : Centrifuge at 300--500g for 5 min at 4 degrees C. Remove supernatant. Resuspend pellet in 1 mL wash buffer (PBS + 1% BSA + RNase inhibitor). Repeat wash once. - Myelin removal (critical for brain): Perform density gradient cleanup using a myelin removal kit or debris removal reagent. This step is typically necessary for white matter-containing samples. - Quality check : Stain with DAPI or Trypan Blue. Count on hemocytometer or automated counter. Inspect morphology: nuclei should appear round, uniform, and debris-free. - Concentration adjustment : Dilute to 700--1,200 nuclei/uL for 10x Genomics loading. #### Expected Results - Nuclei yield : 100,000--225,000 nuclei per mg tissue (cerebellum: avg 192,722 nuclei/mg, SD 29,079) - Quality metrics : Intact nuclei >80%; ambient RNA typically 10--25% (higher without myelin removal) - Processing time : 30--45 min manual; ~7 min on Singulator (plus downstream cleanup) - Downstream compatibility : 10x Chromium 3' and 5', Multiome ATAC+GEX, Parse Biosciences Evercode, Mission Bio Tapestri #### Tips and Tricks - Myelin removal is strongly recommended for brain samples, especially cerebellum and white matter-rich regions. Without it, ambient RNA commonly rises from approximately 10% to 50--60%. Use a density gradient (e.g., Percoll, sucrose cushion) or commercial debris removal reagents. - Lower endogenous RNase activity : Brain tissue has lower RNase activity than many other tissues, but RNase inhibitor is still recommended. Samples may tolerate slightly longer hold times on ice (up to 30--45 min) compared to high-RNase tissues like pancreas or lung. - Frozen mouse brain demo tip : The standard 6--7 min nuclei protocol works for frozen mouse brain. If you observe high debris and damaged nuclei, check that the Singulator was properly pre-cooled and that tissue was not thawed before loading. - Extended protocol for tough brain regions : For fibrous or densely myelinated regions, consider the Extended Nuclei Isolation protocol with longer disruption cycles. - DAPI + FACS sorting : For maximum purity, FACS-sort DAPI-positive nuclei after initial isolation. This typically removes the majority of debris and ambient RNA. - Cerebellum-specific : The Singulator preserves all 12 major cerebellar cell types (granule cells, Purkinje neurons, Bergmann glia, astrocytes, oligodendrocytes, interneurons, etc.) with high reproducibility across biological replicates (Pearson r > 0.99). #### Troubleshooting Problem Possible Cause Solution High debris / myelin contamination Insufficient cleanup Add density gradient or debris removal step; increase wash cycles Low yield from cortex Under-homogenization Increase Dounce strokes to 20--25; check pestle clearance Damaged / lysed nuclei (ghosting) Over-lysis Reduce NP-40 to 0.05%; reduce Dounce strokes; shorten lysis time High ambient RNA (>30%) Myelin debris, over-lysis Add myelin removal; reduce NP-40 concentration; FACS-sort DAPI+ nuclei Nuclei clumping Insufficient MgCl2 or filtration Increase MgCl2 to 5 mM; add 0.04% BSA to wash buffer; filter through 20 um strainer Inconsistent yields between brain regions Variable tissue density Weigh tissue precisely; adjust Dounce strokes per region #### Singulator Protocol Singulator 100 / 200 / 200+ -- Official Singulator Brain Nuclei Isolation Protocol (with myelin removal) Cartridge : NIC+ Nuclei Isolation Cartridge (pre-cooled) Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL); NIC+ Isolation Bundle with RNase Inhibitor V2 (P/N 100-289-1xx) Sample size : 20--100 mg frozen brain tissue Auto-Mince : Yes Incubation : 0 min at Cold (standard) or extended for tough regions Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; myelin removal via density gradient or Nuclei Debris Removal Reagent; filter through 40 um strainer Run time : ~6--7 minutes on instrument Special considerations : Start brain samples first in multi-sample workflows --- brain requires the most downstream processing time for myelin removal Source : Official Singulator brain nuclei protocol; Application Note: Single-Nuclei Sequencing of Mouse Cerebellum Protocol variants used in publications : Extended Nuclei Isolation protocol for 50 mg frozen dlPFC and ACC brain tissue Small-volume nuclei isolation protocol for 3 frozen frontal cortex samples Low Input Nuclei Isolation with modified mixing (Top/Slow) and Dounce disruption (Medium) for ventromedial hypothalamus #### References [1] Romero R, Chu T, González-Robles TJ, et al. The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1. Nature Cancer. 2024;5:1641-1659. DOI: 10.1038/s43018-024-00838-6 [2] Precision Cell Systems. Application Note: Isolation of Mouse Skin Cells. Singulator 100 cell isolation yielding 5,200 cells/mg at 90% viability with scRNA-seq via 10x Genomics Chromium. [3] Nobs SP, et al. A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing. JOVE. 2023;(197):e65611. [https://www.jove.com/t/65611/a-simple-quick-partially-automated-protocol-for-isolation-single](https://www.jove.com/t/65611/a-simple-quick-partially-automated-protocol-for-isolation-single) [4] Haegebarth A, et al. Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies. Nucleus. 2023;14(1):2186686. [https://pmc.ncbi.nlm.nih.gov/articles/PMC10012951/](https://pmc.ncbi.nlm.nih.gov/articles/PMC10012951/) [5] Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) [6] Strobl J, et al. Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse. Science Translational Medicine. 2023;15:eabq0476. [https://www.science.org/doi/10.1126/scitranslmed.abq0476](https://www.science.org/doi/10.1126/scitranslmed.abq0476) [7] Benitez GJ, Shinoda K. Isolation of Adipose Tissue Nuclei for Single-Cell Genomic Applications. Journal of Visualized Experiments. 2020;(160):e61230. [https://pmc.ncbi.nlm.nih.gov/articles/PMC7971773/](https://pmc.ncbi.nlm.nih.gov/articles/PMC7971773/) [8] Drokhlyansky E, et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606-1622. [https://www.sciencedirect.com/science/article/pii/S0092867420309946](https://www.sciencedirect.com/science/article/pii/S0092867420309946) ### C. elegans #### Overview C. elegans is a transparent nematode with 959 somatic cells in adults. The tough cuticle (collagen-based exoskeleton) and small body size make nuclei isolation challenging. Single-nucleus RNA sequencing has been developed to overcome the difficulty of dissociating intact cells from the cuticle. Published protocols involve mechanical disruption (mortar and pestle in liquid nitrogen, or Dounce homogenization) of frozen worm pellets, followed by filtration and FACS sorting. The Singulator has been discussed for C. elegans nuclei isolation. The recommendation is to grind frozen worms into a powder using mortar and pestle in liquid nitrogen, then load the powder into the Singulator cartridge (Precision Cell Systems). #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%), 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, RNase inhibitor - Sample preparation : Freeze worm pellet at -80 degrees C. Grind in liquid nitrogen using mortar and pestle until fine powder. Transfer powder to Singulator cartridge or Dounce homogenizer. - Singulator protocol : Standard Nuclei Isolation V2 with 2--3x disruption cycles (similar to plant tissue protocol approach) - Cartridge : NIC+ - Expected yield : C. elegans has 959 somatic cells; collect large worm pellets (thousands of animals) for adequate nuclei numbers - Key cell types : Neurons (302 in hermaphrodites), muscle cells, intestinal cells, hypodermal cells, germline cells - Tips : Pre-grinding in liquid nitrogen is typically necessary due to the tough cuticle; heavily mince or grind before loading into Singulator; multiple disruption cycles may be needed; FACS sorting recommended to remove cuticle debris #### References [1] Borcherding N, et al. Isolation and RNA sequencing of single nuclei from Drosophila tissues. STAR Protocols. 2022;3(3):101417. [https://www.sciencedirect.com/science/article/pii/S2666166722002970](https://www.sciencedirect.com/science/article/pii/S2666166722002970) ### Cartilage #### Overview Cartilage is an avascular tissue with sparse chondrocytes embedded in a dense extracellular matrix of type II collagen, proteoglycans, and water. The matrix is extremely resistant to mechanical disruption. Enzymatic pre-digestion (Liberase, collagenase) may be needed before nuclei isolation. The Singulator has been used for cartilage processing in the context of synovial membrane/cartilage mixed samples, using 75 ug/mL Liberase for dissociation. #### Recommended Protocol - Pre-treatment : Enzymatic digestion with Liberase (75 ug/mL) or Collagenase II (0.2%) for 30--60 min at 37 degrees C - Lysis buffer : Standard NP-40 (0.1%) on enzymatically softened tissue - Singulator protocol : Extended Nuclei Isolation V2 with pre-enzymatic treatment using Single-Shot mechanism (3 mL enzyme + 3 mL buffer) - Cartridge : NIC+ - Expected yield : Very low (1,000--5,000 nuclei/mg; cartilage has few cells per volume) - Key cell types : Chondrocytes (hyaline, fibrous, or elastic depending on cartilage type) - Tips : Pre-enzymatic treatment is typically necessary for matrix softening; without it, yields are generally very low ### Cochlea #### Overview The cochlea is an extremely small, bony structure containing the organ of Corti with delicate hair cells, supporting cells, spiral ganglion neurons, and stria vascularis. The tissue is encased in the temporal bone and must be carefully dissected. Sample mass is typically sub-milligram for mouse cochlea, requiring the most stringent low-input approaches. #### Recommended Protocol - Sample preparation : Micro-dissect organ of Corti and spiral ganglion from temporal bone under dissecting microscope on cold platform - Lysis buffer : Standard NP-40 (0.05--0.1%) -- gentle formulation for delicate neural tissue - Singulator protocol : Low Volume Nuclei Isolation V2 with NIC+ cartridge - Expected yield : Very low total nuclei; hair cells are rare (~3,400 outer hair cells per human cochlea) - Key cell types : Inner and outer hair cells (very rare), supporting cells, spiral ganglion neurons, stria vascularis cells - Tips : Pool multiple cochleae (4--8 mice) to achieve adequate nuclei numbers; NIC+ cartridge essential for sub-milligram inputs; handle as neural tissue with gentle processing ### Colon / Intestine #### Overview Intestinal tissue has a distinctive architecture with two main layers relevant to nuclei isolation: the epithelial layer (villi and crypts lining the lumen) and the lamina propria (underlying connective tissue containing immune cells, fibroblasts, nerves, and vasculature). Isolating nuclei from whole intestinal tissue captures both compartments simultaneously, which is advantageous for profiling the complete tissue microenvironment. However, the mucus-secreting goblet cells and Paneth cells (in small intestine) release mucus and antimicrobial peptides that can interfere with downstream processing. The Singulator has published use for colon tissue nuclei isolation (MSKCC, Science Translational Medicine) using the Standard Nuclei Isolation protocol with snap-frozen tissue. A Stanford study showed that an alternative Tricine-KOH buffer worked better for intestinal tissues and polyps compared to the standard Singulator buffer. USDA researchers have also used the Singulator for nuclei isolation from pig and cow intestine. #### Recommended Lysis Buffer Standard NP-40 lysis buffer (good starting point): Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation Alternative -- Tricine-KOH buffer (published for intestinal nuclei with Singulator): Component Concentration Notes Tricine-KOH pH 7.8 25 mM Specialized buffer base DTT 1 mM Reducing agent (add fresh) Spermine 0.5 mM Chromatin stabilizer (add fresh) Spermidine 0.5 mM Chromatin stabilizer (add fresh) NP-40 0.1% Lysis detergent RNase inhibitor 0.4 U/uL RNA preservation This buffer has more components that must be added fresh but showed improved performance for intestinal tissues and polyps. #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen intestinal tissue. Remove luminal contents if possible. Weigh 50--100 mg. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat twice. - Filtration : Filter through 40 um strainer. Optional FACS sorting for DAPI+ nuclei. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : Moderate; 20,000--100,000 nuclei per mg depending on intestinal segment and preparation - Cell type composition : Epithelial cells (enterocytes, goblet cells, stem cells, Paneth cells, enteroendocrine cells), immune cells (T cells, B cells, macrophages, ILCs), fibroblasts, smooth muscle, enteric neurons - Quality considerations : Mucus contamination can affect downstream processing - Downstream compatibility : 10x Chromium, 10x Immune Profiling (5' VDJ), RAISIN RNA-seq #### Tips and Tricks - Tricine-KOH buffer for polyps : If processing intestinal polyps, the Tricine-KOH buffer outperforms standard NP-40 buffer. - Singulator open reagent platform advantage : The Singulator Single-Shot mechanism allows loading 3 mL of custom Tricine-KOH buffer in the enzyme slot and 3 mL of wash in the buffer slot for a fully customized run. - EDTA pre-treatment for epithelium : If prioritizing epithelial cell recovery, brief EDTA chelation (2 mM, 15 min, 4 degrees C) before nuclei isolation can help detach epithelial cells from the basement membrane. - FACS sorting recommended : Intestinal nuclei preparations benefit from FACS sorting for DAPI+ nuclei to remove mucus and debris. #### Troubleshooting Problem Possible Cause Solution Mucus contamination Goblet cell contents Additional wash cycles; FACS sort DAPI+ nuclei Low epithelial cell recovery Epithelial cells loosely attached EDTA chelation pre-treatment; gentle processing High debris Food particles / luminal contents Clean tissue thoroughly before processing; remove luminal contents on dry ice Poor RNA quality Enzymatic degradation from Paneth cells Higher RNase inhibitor; rapid processing #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL); or use Single-Shot with custom Tricine-KOH buffer (3 mL enzyme slot + 3 mL buffer slot) Sample size : 50--100 mg frozen intestinal tissue Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; filter through 40 um strainer; optional FACS sorting Run time : ~7 min on instrument Published use : MSKCC used Singulator 100 Standard Nuclei Isolation for snap-frozen human ascending colon, published in Science Translational Medicine (2023); Stanford validated with Tricine-KOH buffer for intestinal tissues and polyps #### References [1] Drokhlyansky E, et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606-1622. [https://www.sciencedirect.com/science/article/pii/S0092867420309946](https://www.sciencedirect.com/science/article/pii/S0092867420309946) [2] Strobl J, et al. Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse. Science Translational Medicine. 2023;15:eabq0476. [https://www.science.org/doi/10.1126/scitranslmed.abq0476](https://www.science.org/doi/10.1126/scitranslmed.abq0476) ### Cornea #### Overview The cornea is a transparent, avascular tissue with five layers: epithelium, Bowman's layer, stroma (keratocytes in organized collagen lamellae), Descemet's membrane, and endothelium. The collagenous stroma is mechanically resistant. Cornea nuclei isolation follows similar principles to skin (collagen-rich, requires Extended protocol). #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 15--20 per pestle - Singulator protocol : Extended Nuclei Isolation V2 - Cartridge : NIC+ (cornea is typically small, 1--5 mg for mouse) - Expected yield : Low (5,000--20,000 nuclei/mg) - Key cell types : Corneal epithelial cells, keratocytes, endothelial cells - Tips : Mince finely; Extended protocol needed for collagenous stroma; pool multiple corneas for mouse samples ### Dorsal Root Ganglia #### Overview Dorsal root ganglia (DRG) are small clusters of sensory neuron cell bodies located along the spinal column. They contain large-diameter sensory neurons, satellite glial cells, and Schwann cells. DRG are small (sub-milligram to a few mg in mice) and contain neurons with very large cell bodies (up to 80 um), making nuclei isolation particularly relevant since these large cells are difficult to capture in microfluidic platforms. #### Recommended Protocol - Sample preparation : Pool multiple DRG per animal; keep frozen until processing - Lysis buffer : Standard NP-40 (0.1%) -- treat as neural tissue with gentle processing - Dounce strokes : 5--10 gentle strokes per pestle - Singulator protocol : Low Volume Nuclei Isolation V2 with NIC+ cartridge - Expected yield : Moderate per mg (sensory ganglia are cellular), but total yield is limited by small tissue mass - Key cell types : Sensory neurons (nociceptors, mechanoreceptors, proprioceptors), satellite glial cells, Schwann cells - Tips : Pool DRG from multiple spinal levels; NIC+ essential for small samples; gentle disruption to preserve large neuron nuclei ### Drosophila #### Overview Drosophila melanogaster presents unique challenges because many cell types are enclosed within the exoskeleton (cuticle), which cannot be dissociated intact using standard methods. This makes single-nucleus RNA sequencing (snRNA-seq) the preferred approach for whole-animal or cuticle-associated tissues (flight muscle, fat body, epidermis). For soft internal tissues (brain, gut, ovary), both scRNA-seq and snRNA-seq are feasible. Published Drosophila snRNA-seq protocols involve mechanical tissue disruption (Dounce homogenization) followed by FACS sorting of nuclei, with libraries prepared via Smart-seq2 or 10x Chromium. UMass Chan Medical School has used the Singulator for Drosophila S2 cell nuclei extraction. The Singulator's automated workflow has been validated for Drosophila cell and nuclei isolation applications. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%), 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 1% BSA, 0.4 U/uL RNase inhibitor - Sample preparation : For whole flies, flash-freeze in liquid nitrogen and grind to powder using mortar and pestle. For dissected tissues (brain, gut), load directly. For cultured cells (S2 cells), centrifuge at 300g for 5 min and resuspend in 100--200 uL NIR. - Singulator protocol : Standard Nuclei Isolation V2; for cultured S2 cells, load cell suspension directly into dissociation chamber - Cartridge : NIC+ (Drosophila tissues are very small) - Expected yield : Variable; brain yields moderate nuclei per mg; whole-fly preparations yield lower quality - Key cell types : Tissue-dependent; CNS neurons, glia, muscle, hemocytes, fat body cells, gut enterocytes - Tips : FACS sorting is strongly recommended for Drosophila nuclei to remove cuticle debris; pool multiple animals for adequate yield; for whole-fly preparations, pre-pulverization in liquid nitrogen is typically necessary #### References [1] Precision Cell Systems. Application Note: Sequencing Lung and Kidney Nuclei: Leveraging the Singulator 100 and Parse Biosciences Evercode Whole Transcriptome v2 Platform. P/N: 100-261-174. ### Embryonic Tissue #### Overview Embryonic tissue is soft, highly cellular, and typically very small in mass -- often well below the standard 50 mg input for most nuclei isolation protocols. The tissue consists of rapidly dividing progenitor cells across all germ layers, with a high nucleus-to-cytoplasm ratio that makes nuclei isolation relatively straightforward from a lysis perspective. However, the small sample size is the primary challenge, requiring the Low Volume or NIC+ cartridge approach on the Singulator. Mouse embryonic brain nuclei isolation has been documented in 10x Genomics protocols, and the Singulator has been used for zebrafish embryos at the University of Oregon. The key considerations are gentle processing (embryonic cells are fragile) and minimizing sample loss from small inputs. #### Recommended Lysis Buffer Standard NP-40 lysis buffer (gentle formulation): Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.05--0.1% Lower detergent for fragile embryonic cells BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, handle embryonic tissue with extreme care. For whole embryos, mince gently. For specific embryonic regions (e.g., brain primordia, limb buds), dissect with cold fine forceps. - Lysis : Add 0.5--1 mL cold lysis buffer (Low Volume). Transfer tissue. - Mechanical disruption : Pestle A, 5--10 gentle strokes. Pestle B, 5--10 gentle strokes. Embryonic tissue disrupts very easily; fewer strokes are needed. - Incubation : Brief (2--3 min) on ice. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend gently. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : High per mg tissue (embryonic tissue is very cellular) - Cell type composition : Diverse progenitor populations, tissue-specific depending on embryonic stage and region - Downstream compatibility : 10x Chromium, 10x Multiome, Smart-seq2 #### Tips and Tricks - NIC+ cartridge is recommended for small samples : Embryonic tissue is often sub-20 mg. The NIC+ cartridge significantly improves nuclei yield from small inputs compared to the standard cartridge (Precision Cell Systems). - Low Volume Nuclei protocol recommended : Reduces reagent volumes and sample loss for small inputs (Precision Cell Systems). - Gentle disruption : Embryonic cells are fragile. Use fewer strokes and slower speeds. Reduce Singulator disruption speed to Medium or Slow. - Multiple embryos per run : Pool multiple embryos (or embryonic regions) to reach adequate tissue mass for reliable isolation. #### Troubleshooting Problem Possible Cause Solution Insufficient tissue mass Single embryo too small Pool multiple embryos; use NIC+ cartridge; use Low Volume protocol Nuclei damage Over-processing of fragile cells Reduce Dounce strokes; reduce Singulator speed Cell type imbalance Developmental stage affects composition Ensure accurate staging; dissect specific regions of interest #### Singulator Protocol Singulator 100 / 200 / 200+ -- Low Volume Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge (essential for small embryonic samples) Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 1--50 mg (NIC+ supports down to 1 mg) Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Medium speed (reduce from Fastest for fragile tissue) Disruption : Default at Medium speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; filter through 40 um strainer Run time : ~7 min on instrument ### Endometrium #### Overview Endometrial tissue consists of a glandular epithelium (columnar cells lining the uterine cavity and glands), a specialized stroma (decidualized fibroblasts), endothelial cells, and a variable immune cell population that fluctuates with the menstrual cycle. The tissue has moderate mechanical resistance and moderate RNase activity. Nuclei isolation from frozen endometrium is straightforward using standard protocols. A validated Singulator nuclei isolation protocol exists for pig endometrium tissue (50--60 mg, Standard Nuclei Isolation, Fastest disruption speed). Human endometrial tissue follows similar processing parameters. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen endometrial tissue. Weigh 50--60 mg. - Lysis : Add 1 mL cold lysis buffer. Transfer tissue to Dounce homogenizer. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : Moderate to high; 30,000--100,000 nuclei per mg depending on cycle phase and tissue composition - Cell type composition : Glandular epithelial cells, stromal fibroblasts (decidualized in secretory phase), endothelial cells, uNK cells, macrophages, T cells - Downstream compatibility : 10x Chromium, 10x Multiome #### Tips and Tricks - Pre-cool the Singulator for approximately 15 min prior to loading (Precision Cell Systems). - Standard protocol works well : Endometrium is moderately soft; Standard Nuclei Isolation at Fastest speed is effective (Precision Cell Systems). - Post-processing : Two centrifugation-resuspension cycles are recommended, with final resuspension in 250--500 uL NSR + 0.4 U/uL RNase inhibitor (Precision Cell Systems). #### Troubleshooting Problem Possible Cause Solution Mucus contamination Secretory epithelium Additional wash cycles; FACS sort DAPI+ nuclei Low yield Insufficient tissue mass Use NIC+ cartridge for sub-30 mg samples Variable composition Menstrual cycle stage Document cycle phase; this affects cell type proportions #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge (or standard blue cartridge for 50+ mg) Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 50--60 mg frozen endometrial tissue Pre-cooling : Cool Singulator for approximately 15 min before loading (Precision Cell Systems) Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in 500 uL NSR + 0.4 U/uL RNase inhibitor; repeat centrifugation; final resuspension in 250 uL NSR + 0.4 U/uL RNase inhibitor (Precision Cell Systems) Run time : ~7 min on instrument ### Esophagus #### Overview Esophagus has a stratified squamous epithelium (non-keratinized), lamina propria, muscularis mucosae, submucosa, and muscularis propria (skeletal muscle in upper third, smooth muscle in lower third). The squamous epithelium is moderately resistant to lysis. Standard to Extended Nuclei Isolation protocols are appropriate. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 10--15 per pestle - Singulator protocol : Standard or Extended Nuclei Isolation V2 - Cartridge : NIC+ for biopsies - Expected yield : Moderate (20,000--60,000 nuclei/mg) - Key cell types : Squamous epithelial cells, smooth muscle cells, fibroblasts, immune cells, enteric neurons - Tips : Process similarly to skin (squamous epithelium) but somewhat easier; Standard protocol may be sufficient ### FFPE Tissue #### Overview Formalin-fixed, paraffin-embedded (FFPE) tissue represents the vast majority of archived clinical specimens in biobanks worldwide. Nuclei isolation from FFPE requires deparaffinization (removal of paraffin wax), rehydration, and gentle mechanical disruption. The formalin fixation cross-links proteins and nucleic acids, making RNA extraction more challenging than from fresh/frozen tissue. Despite this, FFPE snRNA-seq is increasingly feasible with modern platforms such as 10x Genomics Flex Assay. CRITICAL : FFPE tissue processing on the Singulator is exclusive to the Singulator 200+ . The Singulator 100 and Singulator 200 do not support FFPE processing (Precision Cell Systems). #### Singulator 200+ FFPE Workflow The Singulator 200+ provides a fully automated, two-step cartridge workflow for FFPE nuclei isolation: Step 1 -- FFPE Cartridge (GREEN) : Automated deparaffinization and rehydration - 50 um FFPE curl loaded into FFPE cartridge - Uses a safe, proprietary solvent (no fume hood required, no toxic solvents like CitriSolv) - Automated rehydration through ethanol series and PBS rinse - Duration: approximately 40 minutes (Precision Cell Systems) Step 2 -- NIC+ Cartridge (YELLOW) : Transfer tissue to NIC+ cartridge - Add 75 uL RNase Inhibitor V2 - Run the 19-minute FFPE Nuclei Isolation protocol - Automated nuclei isolation from processed tissue (Precision Cell Systems) #### Performance Data Metric Singulator 200+ Manual (Miltenyi) Total time ~1 hour ~2 hours Hands-on time 25 minutes Pipetting steps 4 28 Erythrocyte contamination 1% 5% Toxic solvents required No Yes (CitriSolv, fume hood) Source: Precision Cell Systems Application Note: FFPE Tissue Dissociation #### Validated FFPE Applications - Mouse pancreatic ductal adenocarcinoma (PDAC) -- published application note with 10x Flex Assay (Precision Cell Systems) - Mouse brain with melanoma metastasis (MSKCC researchers) -- integrated with 10x Xenium spatial platform (Precision Cell Systems) - PERF-seq rare cell sequencing from FFPE (Stanford/MSKCC collaboration) (Precision Cell Systems) #### Tips and Tricks - Input requirements : Minimum 2 mg tissue; compatible with 50 um FFPE curls (Precision Cell Systems) - Enriches fragile cell types : The Singulator 200+ FFPE workflow enriches for ductal cancer cells and cancer-associated fibroblasts compared to manual methods, which skew toward immune cells (Precision Cell Systems) - Chromatin accessibility caution : FFPE nuclei processed on the Singulator may show downstream issues with ATAC-seq and ChIP-seq despite good nuclei morphology. Library and sequencing failures have been reported for epigenomic applications. If chromatin integrity is critical, consider lowering RPM and using shorter programs (Precision Cell Systems) - Not for S100 or S200 : FFPE processing is exclusive to the Singulator 200+ (Precision Cell Systems) #### Alternative Manual FFPE Protocol (for comparison) - Deparaffinization: 3 cycles of 10 min in CitriSolv (requires fume hood) - Manual rehydration through ethanol series (100%, 100%, 70%, 50%, 30%) - PBS washes (2x) - Enzyme digestion: 45 min in 1 mg/mL Liberase - Mechanical dissociation (Miltenyi OctoMACS or equivalent) - Centrifugation, filtration, counting This manual process requires 2+ hours, 28 pipetting steps, and toxic solvents, compared to ### Heart #### Overview Heart tissue presents unique challenges for nuclei isolation. Cardiomyocytes constitute approximately 30--40% of cardiac cells by number but 70--80% by volume, and they are among the largest mammalian cells (50--150 um). These cells are virtually impossible to capture intact in standard single-cell microfluidic platforms, making nuclei isolation the preferred approach for cardiac transcriptomics. Additional challenges include high extracellular matrix content (collagen, elastin) and dense connective tissue. #### Recommended Lysis Buffer Standard NP-40 lysis buffer with possible speed reduction for gentle nuclei release: Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.2 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : Mince frozen or fresh heart tissue roughly (not super fine) on dry ice using a clean blade. Aim for 2--3 mm pieces. For Singulator, the Standard Nuclei Isolation protocol is recommended with rough mincing before loading. - Lysis : Add 1 mL cold lysis buffer to a pre-chilled 2 mL Dounce homogenizer. Transfer minced tissue. - Mechanical disruption : Pestle A, 15--20 strokes (heart tissue is tough, requires more strokes). Pestle B, 15--20 strokes. - Washing : Centrifuge at 500g for 5 min at 4 degrees C. Resuspend in 1 mL wash buffer. Repeat. - Filtration : Filter through 40 um strainer. - Debris removal (recommended): Heart tissue produces significant connective tissue debris. Density gradient cleanup or debris removal reagent is recommended. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : 10,000--30,000 nuclei per mg tissue (variable; see note below) - Quality metrics : Nuclei should be round and uniform; expect some connective tissue debris - Processing time : 30--40 min manual; ~7 min on Singulator - Downstream compatibility : 10x Chromium, Parse Biosciences, BioSpyder TempO-LINC Note on yield variability : Data show heart nuclei yields ranging from 10,000--13,000 to approximately 30,000 nuclei/mg depending on tissue region, mouse strain, and protocol parameters. The lower range (10--13K/mg) appears more typical for standard protocols; higher yields (30K/mg) may reflect optimized conditions or different tissue regions. #### Tips and Tricks - Rough mincing, not fine : Rough-mince heart tissue before processing. Fine mincing can damage the connective tissue matrix in ways that increase debris without improving nuclei yield. - Speed adjustment : If nuclei appear damaged, reduce disruption speed (on Singulator) or Dounce strokes. Heart tissue benefits from slightly slower processing than brain. - Large Cell Cartridge : For Singulator heart nuclei isolation, the Large Cell Isolation Cartridge (P/N 100-258-668) may be required depending on protocol. - Mouse Heart Reagent : Precision Cell Systems developed a specific Mouse Heart Reagent (P/N 100-246-863) containing Percoll Stock Solution for nuclei cleanup. - DMEM without Ca/Mg : Use DMEM without calcium and magnesium for reconstitution buffers when performing heart nuclei isolation with enzymatic pre-treatment. #### Troubleshooting Problem Possible Cause Solution Low yield ( Tough connective tissue Increase Dounce strokes; try Extended protocol; pre-mince more thoroughly High debris Collagen/elastin fibers Add density gradient cleanup; use Nuclei Debris Removal Reagent Damaged nuclei Over-processing Reduce speed/strokes; use gentle lysis buffer (TST) Poor pellet formation Low nuclei density Increase centrifuge speed to 700g for 5 min #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge or Large Cell Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL); optional Mouse Heart Reagent with Percoll Sample preparation : Rough-mince heart tissue on dry ice (not fine mince) Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed (reduce to Slow if nuclei appear damaged) Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR; density gradient or debris removal recommended Run time : ~7 minutes Source : Precision Cell Systems protocols; Mouse Heart Enzyme beta (P/N 100-247-099) #### References [1] Protocols.io. Nuclei Isolation for SnRNA-seq and SnATAC-seq from Frozen Fresh Human Retina Sample. [https://www.protocols.io/view/nuclei-isolation-for-snrna-seq-and-snatac-seq-from-bftmjnk6](https://www.protocols.io/view/nuclei-isolation-for-snrna-seq-and-snatac-seq-from-bftmjnk6) [2] Lukowski SW, et al. Single-nuclei RNA-seq on human retinal tissue provides improved transcriptome profiling. Nature Communications. 2019;10:5743. [https://www.nature.com/articles/s41467-019-12917-9](https://www.nature.com/articles/s41467-019-12917-9) ### iPSC-Derived Tissues #### Overview iPSC-derived tissues (cardiomyocytes, neurons, hepatocytes, organoids, etc.) grown in culture are processed similarly to organoids and cell suspensions. These samples are typically available as cell pellets or as 2D/3D culture constructs. Nuclei isolation follows the cell culture nuclei protocol: centrifuge to pellet, resuspend in NIR, and load into the Singulator. #### Recommended Protocol - Sample preparation : Aspirate culture medium; wash with cold PBS; detach cells from dish (accutase or TrypLE for monolayers; dissolve Matrigel for 3D cultures); centrifuge 300g, 5 min; resuspend pellet in 100--200 uL NIR. - Lysis buffer : NIR (Singulator's nuclei isolation reagent) or standard NP-40 (0.1%) - Singulator protocol : Standard Nuclei Isolation V2 - Cartridge : NIC+ (iPSC-derived samples are often small) - Expected yield : High (cultured cells are densely packed); 50,000--500,000 nuclei per million cells - Key cell types : Depends on differentiation protocol (cardiomyocytes, neurons, hepatocytes, etc.) - Tips : Gentle disruption settings; iPSC-derived cells are often more fragile than primary tissue; for 2D cultures, centrifuge first, then load as pellet in NIR ### Kidney #### Overview Kidney is one of the most tractable tissues for nuclei isolation. It has moderate RNase activity, yields well, and the resulting nuclei represent the full spectrum of renal cell types (proximal tubular cells, distal tubular cells, collecting duct cells, podocytes, endothelial cells, immune cells, interstitial cells). Nuclei isolation is particularly useful for kidney because some cell types (e.g., podocytes, large tubular epithelial cells) are underrepresented in enzymatic dissociations. The tissue has relatively low endogenous RNase activity, allowing slightly longer handling times. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.2 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : Mince frozen or fresh kidney tissue on dry ice. Weigh 50--100 mg. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining, counting, morphology inspection. #### Expected Results - Nuclei yield : 15,300,000 total from 100 mg tissue on Singulator (approximately 153,000 nuclei/mg) - Quality metrics : Kidney nuclei median genes per cell: 2,634; median transcripts per cell: 6,935 (with Parse Biosciences platform) - Processing time : 20--30 min manual; ~7 min Singulator plus 15 min wash/RBC lysis - Downstream compatibility : 10x Chromium, Parse Biosciences Evercode, SPLiT-seq #### Tips and Tricks - Low RNase tissue : Kidney has low endogenous RNase activity, so samples can tolerate slightly longer hold times on ice (similar to brain). - Standard nuclei protocol works well : For the Singulator, the standard nuclei isolation protocol is recommended for kidney. No specialized enzyme or extended protocol is needed. - RBC lysis : Kidney is vascular; include an RBC lysis step (ACK buffer, 3 min on ice) after initial centrifugation if the nuclei pellet appears red. - NIC+ for small samples : For biopsies or sub-20 mg samples, the NIC+ cartridge yields significantly more nuclei than the standard Nuclei Isolation Cartridge. Mean yield from NIC+: 158,696 nuclei from 1--10 mg mouse kidney (vs. 119,699 from standard cartridge). - Batch processing for large studies : For studies requiring 300+ samples (e.g., SPLiT-seq), process 8 samples at a time through nuclei isolation, then batch-fix and freeze for later barcoding. #### Troubleshooting Problem Possible Cause Solution Red pellet RBC contamination Add ACK lysis step (3 min on ice) after first centrifuge Low yield from biopsy Insufficient tissue Use NIC+ cartridge; ensure tissue is loaded into disruption chamber High ambient RNA Debris carryover Add additional wash cycle; consider density gradient cleanup Clumping Insufficient filtration Use sequential filtration (70 um then 40 um then 20 um) #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol (or Low Volume Nuclei for small samples) Cartridge : NIC+ Nuclei Isolation Cartridge (standard or for sub-20 mg samples) Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL); 75 uL Protector RNase Inhibitor added directly to dissociation chamber Sample size : 1--100 mg kidney tissue Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; RBC lysis if needed; filter through 40 um strainer Run time : ~7 minutes on instrument, plus ~15 min wash/RBC lysis Yield data : 15.3M nuclei from 100 mg mouse kidney (NIC+ cartridge); 153,000 nuclei/mg; 158,696 total nuclei from sub-20 mg on NIC+ Source : Official Singulator frozen tissue nuclei protocol; Application Note: Sequencing Lung and Kidney Nuclei #### References [1] Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) [2] Precision Cell Systems. Application Note: Sequencing Lung and Kidney Nuclei: Leveraging the Singulator 100 and Parse Biosciences Evercode Whole Transcriptome v2 Platform. P/N: 100-261-174. [3] Borcherding N, et al. Isolation and RNA sequencing of single nuclei from Drosophila tissues. STAR Protocols. 2022;3(3):101417. [https://www.sciencedirect.com/science/article/pii/S2666166722002970](https://www.sciencedirect.com/science/article/pii/S2666166722002970) [4] Precision Cell Systems. Application Note: Use of NIC+ with the Singulator 100 for Sub-20 mg Samples. NIC+ vs Standard cartridge yields across brain, kidney, lung, heart, breast tissues. ### Liver #### Overview Liver tissue is metabolically active with high protein content, making it amenable to nuclei isolation but with important caveats. Hepatocytes are large (20--40 um) polyploid cells that dominate liver parenchyma by volume but are fragile and prone to lysis during dissociation. Nuclei isolation from liver preferentially captures non-parenchymal cells (endothelial cells, Kupffer cells, stellate cells, cholangiocytes) at higher proportions than their true in vivo abundance, with hepatocyte nuclei representing approximately 20% of the captured population and showing 60--70% viability. The Singulator produces almost twice as many nuclei from liver compared to manual methods, though nuclei may be "leakier" (more ambient RNA). Key consideration : If hepatocytes are the primary target, cell isolation with collagenase perfusion may be more appropriate, though nuclei isolation captures the full cellular landscape including rare populations. #### Recommended Lysis Buffer Standard NP-40 lysis buffer --- liver is well-suited to the standard formulation: Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.2 U/uL RNA preservation Alternative : Nuclei EZ Prep (Sigma) has been used successfully for liver nuclei isolation. #### Step-by-Step Protocol - Tissue preparation : Mince frozen liver tissue on dry ice. Weigh 30--60 mg. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. Liver is relatively soft and homogenizes easily. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat. - RBC lysis (recommended): Liver is highly vascular. Add ACK lysis buffer if pellet is red. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining, counting. Expect bimodal size distribution (small non-parenchymal nuclei + large hepatocyte nuclei). #### Expected Results - Nuclei yield : Singulator produces approximately twice the nuclei yield compared to manual Dounce for liver, though nuclei may show higher ambient RNA - Cell type composition : Approximately 60--80% non-parenchymal nuclei (endothelial, Kupffer, stellate); approximately 20% hepatocyte nuclei - Hepatocyte viability : 60--70% for hepatocyte-derived nuclei - Processing time : 25 min on Singulator plus 15 min wash/RBC lysis - Downstream compatibility : 10x Chromium, snRNA-seq, ATAC-seq #### Tips and Tricks - Slowest speed on Singulator : For liver nuclei on the Singulator, modify the dissociation speed to the Slowest setting. This preserves hepatocyte nuclei integrity. - Expect non-parenchymal enrichment : The Singulator preferentially isolates non-parenchymal cells from liver. Set expectations accordingly in experimental design. - Mouse liver snRNA-seq QC : When analyzing liver snRNA-seq data, check for resolution issues in clustering. Ensure adequate sequencing depth and appropriate resolution parameters in Seurat to distinguish hepatocyte subpopulations from non-parenchymal cells. - Debris removal helps liver : Liver produces significant lipid debris. The Nuclei Debris Removal Reagent improves sequencing metrics. - RBC contamination is common : Liver is one of the most vascular organs. Always include an ACK lysis step. #### Troubleshooting Problem Possible Cause Solution Low hepatocyte representation Fragile large nuclei lysing during processing Reduce disruption speed; use Slowest setting on Singulator High ambient RNA Leaky nuclei from aggressive lysis Reduce NP-40; add debris removal step; use Nuclei Debris Removal Reagent Red pellet Heavy RBC contamination ACK lysis, 3--4 min on ice; repeat if needed Lipid debris layer Liver fat content Aspirate floating debris carefully; add density gradient cleanup Discordant cell type proportions Non-parenchymal enrichment Expected behavior; account for in analysis with cell type deconvolution #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol (Slowest Speed) Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 30--60 mg frozen liver Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Slowest speed (critical for hepatocyte nuclei preservation) Post-processing : Centrifuge 500g, 5 min, 4 degrees C; RBC lysis with ACK buffer; resuspend in NSR; Nuclei Debris Removal Reagent (P/N 100-261-392); filter through 40 um strainer Run time : ~7 min on instrument plus ~15 min wash/RBC lysis NIC+ yield comparison : 58,633 nuclei total (NIC+ cartridge) vs. 34,680 (standard cartridge) for sub-20 mg samples Source : Official Singulator frozen tissue nuclei protocol; Application Note: Use of Nuclei Debris Removal Reagent #### References [1] Precision Cell Systems. Application Note: Use of the Nuclei Debris Removal Stock Reagent. P/N: 100-261-392. [2] Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) [3] Mazutis L, Masilionis I, Chaudhary O. Frozen tissue dissociation for single-nucleus RNA-Seq. protocols.io. [https://www.protocols.io/view/frozen-tissue-dissociation-for-single-nucleus-rna-5k5g4y6](https://www.protocols.io/view/frozen-tissue-dissociation-for-single-nucleus-rna-5k5g4y6) [4] Nadelmann ER, et al. Isolation of Nuclei from Mammalian Cells and Tissues for Single-Nucleus Molecular Profiling. Current Protocols. 2021;1(5):e132. [https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.132](https://currentprotocols.onlinelibrary.wiley.com/doi/10.1002/cpz1.132) [5] Precision Cell Systems. Application Note: Use of NIC+ with the Singulator 100 for Sub-20 mg Samples. NIC+ vs Standard cartridge yields across brain, kidney, lung, heart, breast tissues. ### Lung #### Overview Lung tissue is complex, with more than 40 distinct cell types spanning epithelial (AT1, AT2, club cells, ciliated cells, basal cells), endothelial, mesenchymal, and immune compartments. Nuclei isolation is valuable for lung because it captures large pneumocytes and enables profiling of frozen clinical specimens. However, lung has moderately high endogenous RNase activity, requiring careful RNA preservation. Lung nuclei yields are generally lower than kidney or brain per mg tissue because of the tissue's low cellularity (air-filled alveolar spaces). A significant concern with lung nuclei is ambient RNA. In one optimization study, lung ambient RNA averaged 51.6% and increased to 61.2% when disruption intensity was reduced (half-grind protocol), indicating that disruption intensity must be carefully balanced. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4--1.0 U/uL Higher concentration needed due to RNase activity Alternative for frozen lung : MSKCC lysis buffer with DEPC has been validated on frozen human lung tissue. #### Step-by-Step Protocol - Tissue preparation : Mince frozen lung tissue on dry ice. Weigh 50--109 mg. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. Full-grind protocol is preferred over half-grind to minimize ambient RNA. - Incubation (optional): Brief 5 min incubation on ice may improve yield from dense lung tissue. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat twice. - Filtration : Filter through 35--40 um strainer. - FACS sorting (optional but recommended): Sort DAPI-positive nuclei to maximize purity. Validated for frozen lung by MSKCC. - Quality check : DAPI staining, counting. Inspect for debris. #### Expected Results - Nuclei yield : 3,550,000 from 109 mg mouse lung on Singulator (approximately 32,500 nuclei/mg); lower than kidney due to air space - Quality metrics : Lung nuclei median genes per cell: 1,737; median transcripts per cell: 3,633 (Parse Biosciences platform) - Ambient RNA : 50--60% typical without FACS sorting; significantly lower with DAPI+ sorting - Processing time : 45 min on Singulator; 30--40 min manual - Downstream compatibility : 10x Chromium, Parse Biosciences Evercode, inDrops #### Tips and Tricks - Higher RNase inhibitor : Lung has higher endogenous RNase activity than brain or kidney. Use 0.4--1.0 U/uL RNase inhibitor in all buffers. - Do not reduce disruption intensity : Reducing grinding cycles increases ambient RNA from approximately 52% to 61%. Use full disruption protocol. - Process promptly : Lung nuclei do not tolerate long hold times. Process within 30 minutes of lysis. - Frozen tissue yields more : Frozen lung tissues have higher nuclei yields overall compared to fresh tissue. - FACS sorting strongly recommended : Lung nuclei preparations contain significant debris from alveolar structures. FACS sorting for DAPI+ nuclei dramatically improves downstream data quality. #### Troubleshooting Problem Possible Cause Solution Very high ambient RNA (>50%) Intrinsic to lung tissue FACS-sort DAPI+ nuclei; do not reduce disruption intensity Low yield Air-filled tissue, low cellularity Use more starting material (100+ mg); ensure tissue pieces are dense, not air-filled RNase degradation High RNase activity Increase RNase inhibitor to 1.0 U/uL; work quickly; keep on ice at all times Immune cell dominance Inflammatory infiltrate Expected; gate computationally in analysis #### Singulator Protocol Singulator 100 / 200 / 200+ -- Low Volume Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge (pre-cooled) Reagents : NSR + NIR + 75 uL Protector RNase Inhibitor added directly to dissociation chamber Sample size : 50--109 mg lung tissue Auto-Mince : Yes Incubation : 10 min at Cold (Low Volume Nuclei protocol) Mixing : Top at Fast speed Disruption : Default at Fast speed Post-processing : Remove nuclei suspension with 1 mL pipette; centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; filter through 40 um strainer; optional FACS sorting for DAPI+ nuclei Run time : ~7 min on instrument Yield data : 3.55M nuclei from 109 mg mouse lung; 14,097 nuclei captured in sequencing Source : Official Singulator frozen tissue nuclei protocol; Application Note: Sequencing Lung and Kidney Nuclei #### References [1] Precision Cell Systems. Application Note: Sequencing Lung and Kidney Nuclei: Leveraging the Singulator 100 and Parse Biosciences Evercode Whole Transcriptome v2 Platform. P/N: 100-261-174. [2] Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) ### Lymph Node #### Overview Lymph nodes are small, encapsulated lymphoid organs with a well-defined cortex (B cell follicles, germinal centers) and paracortex (T cell zone). Like spleen, lymph nodes are highly cellular and relatively soft, making nuclei isolation straightforward. The primary cell types are B cells, T cells, dendritic cells, macrophages, follicular dendritic cells, and stromal reticular cells. The capsule and trabeculae provide some structural resistance, but once breached, the parenchyma disrupts easily. The Singulator has been used for lymph node tissue processing by USDA researchers, including pig and cow lymph nodes. The Standard Nuclei Isolation protocol is appropriate for this soft, highly cellular tissue. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis BSA 1% Carrier protein RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, bisect or quarter frozen lymph node. Remove capsule if accessible. Weigh 20--60 mg. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : High; lymph nodes are very cellular (50,000--200,000 nuclei per mg) - Cell type composition : B cells (predominant in cortex), T cells (predominant in paracortex), dendritic cells, macrophages, follicular dendritic cells, stromal cells - Downstream compatibility : 10x Chromium, 10x Immune Profiling (5' VDJ), BD Rhapsody, CITE-seq #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 20--60 mg Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Run time : ~7 min on instrument ### Muscle (Skeletal) #### Overview Skeletal muscle is structurally unique: muscle fibers (myofibers) are multinucleated syncytia that can be centimeters long and contain hundreds of myonuclei. These fibers are too large for any microfluidic platform, making nuclei isolation essential for skeletal muscle transcriptomics. Additional cell types include satellite cells (muscle stem cells), fibro-adipogenic progenitors (FAPs), endothelial cells, pericytes, immune cells, and Schwann cells. The dense, fibrous extracellular matrix (endomysium, perimysium, epimysium) requires vigorous mechanical disruption. #### Recommended Lysis Buffer Modified NP-40 lysis buffer with potentially higher detergent for tough connective tissue: Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1--0.2% May need higher concentration for muscle Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.2--0.4 U/uL RNA preservation Published alternative : The Petrany et al. (2020) protocol uses 0.1% NP-40 with 10 mM Tris, 10 mM NaCl, 3 mM MgCl2, and 0.1% Tween-20. #### Step-by-Step Protocol - Tissue preparation : Mince frozen skeletal muscle on dry ice with clean blade. Muscle is very fibrous; mince as finely as possible. Weigh 50--100 mg. - Optional enzymatic pre-treatment : Some protocols use brief Collagenase B + Dispase II treatment (37 degrees C, 15--30 min) before nuclei isolation to soften connective tissue. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 15--20 strokes (muscle requires more strokes). Pestle B, 15--20 strokes. Tissue should be fully homogenized. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat twice. - Filtration : Sequential filtration through 70 um then 40 um strainers to remove connective tissue fragments. - Quality check : DAPI staining. Muscle myonuclei are distinctly elongated compared to round satellite cell nuclei. #### Expected Results - Nuclei yield : Variable; 10,000--50,000 nuclei per mg depending on tissue quality and disruption intensity - Cell type composition : Myonuclei (60--70%), FAPs, satellite cells, endothelial cells, immune cells - Processing time : 40--60 min manual; ~7 min Singulator plus downstream processing - Downstream compatibility : 10x Chromium, Multiome ATAC+GEX #### Tips and Tricks - Muscle is tough : Requires more Dounce strokes than most tissues. Under-homogenization is the most common failure mode. - NIC+ cartridge for biopsies : Human muscle biopsies are small; use the NIC+ cartridge on the Singulator. - Myonuclei identification : Myonuclei are transcriptionally distinct from other cell types and can be identified by markers such as TTN, MYH1/2, and MYBPC1. - Collagenase B + Dispase II pre-treatment : Published protocols use Collagenase B and Dispase II (not Collagenase II) for connective tissue softening. - Avoid extended lysis : Muscle nuclei are susceptible to over-lysis. Keep total lysis + disruption time under 10 min. #### Troubleshooting Problem Possible Cause Solution Low yield Insufficient disruption of fibrous tissue Increase Dounce strokes (20--25 per pestle); consider enzymatic pre-treatment Connective tissue debris Residual fibers Add sequential filtration (70 um then 40 um then 20 um) Myofiber clumps Incomplete mincing Mince more finely; use scissors on dry ice before loading Satellite cell loss Over-processing Use gentler disruption; satellite cells are small and fragile #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge (for standard samples); NIC+ required for human biopsies Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 10--100 mg (NIC+ for sub-20 mg biopsies) Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR; filter through 40 um strainer Run time : ~7 minutes Source : Standard Nuclei Isolation protocol #### References [1] Petrany MJ, et al. Single-nucleus RNA-seq identifies transcriptional heterogeneity in multinucleated skeletal myofibers. Nature Communications. 2020;11:6374. ### Organoids #### Overview Organoids are three-dimensional, self-organizing tissue cultures derived from stem cells or tissue progenitors. They range from simple single-cell-type spheroids to complex multi-lineage structures mimicking intestine, brain, liver, kidney, or tumor microenvironments. Organoids are typically small (sub-millimeter to a few millimeters), grown in Matrigel or similar extracellular matrix, and can be fresh or cryopreserved. Nuclei isolation from organoids requires gentle processing to avoid over-disrupting the already loosely organized structures. The Singulator has been used for organoid nuclei isolation by multiple research groups. The general approach is to resuspend the organoid pellet in 100--200 uL NIR and load directly into the Singulator dissociation chamber. #### Recommended Protocol - Sample preparation : For fresh organoids, aspirate medium; for Matrigel-embedded organoids, dissolve Matrigel with cold Cell Recovery Solution or cold PBS for 30--60 min on ice; centrifuge 300g, 5 min to collect organoids. For snap-frozen organoid pellets, proceed directly. - Lysis buffer : Standard NP-40 (0.1%), gentle formulation with BSA and RNase inhibitor - Singulator protocol : Standard Nuclei Isolation V2; resuspend organoid pellet in 100--200 uL NIR and load directly into dissociation chamber - Cartridge : NIC+ (organoid samples are typically small) - Expected yield : Variable; depends on organoid type, size, and number; pool at least 4 organoids to reduce heterogeneity - Key cell types : Depends on organoid type (e.g., intestinal organoids: enterocytes, goblet cells, Paneth cells, stem cells; brain organoids: neurons, glia, progenitors) - Tips : Use at least 4 organoids per extraction for brain organoids to reduce batch effects; the buffer with pellet (up to 400 uL) can be loaded directly onto the cartridge (Precision Cell Systems); NIC+ cartridge for all organoid samples; gentle disruption settings #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample preparation : Resuspend organoid pellet in 100--200 uL NIR; load buffer + pellet directly into dissociation chamber (Precision Cell Systems) Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Medium speed (organoids are fragile) Disruption : Default at Medium speed Run time : ~7 min on instrument ### Ovary #### Overview Ovarian tissue has a complex architecture consisting of the cortex (containing follicles at various stages, oocytes, granulosa cells, and theca cells) and the medulla (vascular connective tissue). The ovarian stroma is dense and fibrous, particularly in the cortex, which creates moderate challenges for mechanical disruption. The tissue composition varies dramatically with reproductive stage and between species. The Singulator has been demonstrated with zebrafish ovary tissue at the University of Oregon using papain/spleen enzyme formulations for cell isolation. For nuclei isolation from frozen mammalian ovary, the Extended Nuclei Isolation protocol is recommended due to the fibrous cortical stroma. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen ovarian tissue as finely as possible. The cortical stroma is firm; use a sharp blade. Weigh 20--60 mg. - Lysis : Add 1 mL cold lysis buffer. Transfer to Dounce homogenizer. - Mechanical disruption : Pestle A, 15--20 strokes. Pestle B, 15--20 strokes. - Incubation : 5 min on ice (Extended protocol recommended). - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend. Repeat twice. - Filtration : Filter through 70 um then 40 um strainers. - Quality check : DAPI staining. #### Expected Results - Nuclei yield : Moderate; 10,000--50,000 nuclei per mg depending on tissue composition and follicular content - Cell type composition : Granulosa cells, theca cells, stromal fibroblasts, endothelial cells, immune cells, oocyte nuclei (germinal vesicles, rare) - Downstream compatibility : 10x Chromium, 10x Multiome #### Singulator Protocol Singulator 100 / 200 / 200+ -- Extended Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 20--60 mg frozen ovarian tissue Auto-Mince : Yes Incubation : 5 min at Cold (Extended) Mixing : Top at Fastest speed Disruption : Default at Fastest speed, dual disruption cycle Run time : ~12 min on instrument ### Pancreas #### Overview Pancreatic tissue presents unique challenges for nuclei isolation due to its extremely high endogenous RNase and protease activity. The exocrine compartment (acinar and ductal cells) dominates the tissue by mass and contains concentrated digestive enzymes (trypsinogen, chymotrypsinogen, elastase, lipase) stored in zymogen granules. When these cells lyse during nuclei isolation, the released enzymes rapidly degrade RNA and proteins, making pancreas one of the most technically demanding tissues for snRNA-seq. The endocrine compartment (islets of Langerhans, comprising alpha, beta, delta, PP, and epsilon cells) constitutes only 1--2% of total pancreatic mass but is frequently the primary target for diabetes and metabolic research. Pancreatic ductal adenocarcinoma (PDAC) research adds an additional layer of complexity due to the dense desmoplastic stroma that surrounds tumor cells. Despite these challenges, successful snRNA-seq has been performed on both normal and cancerous pancreatic tissue using the Singulator and manual methods. The key to success is aggressive RNase inhibition and rapid processing. #### Recommended Lysis Buffer Citric acid-based buffer (preferred for pancreas per recent publications): Component Concentration Notes Citric acid 0.1 M Acidic pH inhibits RNases; optimal for pancreas Tween-20 0.1% Gentle detergent BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 1.0 U/uL Maximum concentration essential Alternative -- Standard NP-40 lysis buffer with maximal RNase protection : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 1.0 U/uL Maximum concentration -- pancreas requires the highest level VRC (optional) 10 mM Vanadyl ribonucleoside complex provides additional RNase inhibition #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen pancreatic tissue with a clean blade into sub-millimeter pieces. Weigh 26--200 mg. - Lysis : Add 1 mL cold lysis buffer (citric acid- or NP-40-based) to Dounce homogenizer. Transfer tissue immediately. - Mechanical disruption : Pestle A (loose), 10--15 gentle strokes. Pestle B (tight), 10--15 strokes. Pancreas is soft and homogenizes quickly; do not over-disrupt. - Incubation : 5 min on ice in lysis buffer to complete nuclear release. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer containing 1.0 U/uL RNase inhibitor. Repeat wash twice. - Filtration : Filter through 40 um strainer. Optional second filtration through 20 um strainer. - Sucrose gradient purification (recommended for PDAC samples): Layer nuclei suspension over 1.8 M sucrose cushion. Centrifuge 13,000g, 45 min, 4 degrees C. Collect nuclei pellet. - Quality check : DAPI staining and counting. #### Expected Results - Nuclei yield : Highly variable (10,000--100,000 nuclei/mg) depending on tissue composition and preservation - Cell type composition : Acinar nuclei dominate (>60%), with ductal, endocrine (islet), stellate, and immune populations - RNA quality : Challenging; DV200 values typically 70--85% with proper RNase inhibition - Processing time : 45--60 min manual; Singulator nuclei protocol is ~7 min on instrument plus downstream processing - Downstream compatibility : 10x Chromium, Mission Bio Tapestri (snDNA-seq validated), 10x Flex Assay #### Tips and Tricks - Maximal RNase inhibition is non-negotiable : Pancreas has higher RNase expression than most tissues. Use 1.0 U/uL RNase inhibitor in all buffers. - Citric acid buffer outperforms NP-40 for archival pancreas : A 2024 comparison of five nuclei isolation protocols found the citric acid method produced the highest RNA integrity and lowest cytoplasmic contamination from frozen archival human pancreata. - Process quickly : Minimize time between lysis and downstream application. Every additional minute degrades RNA in pancreas tissue. - VRC supplementation : Adding 10 mM vanadyl ribonucleoside complex alongside standard RNase inhibitor provides an additional layer of RNA protection. - Sucrose gradient for tumors : PDAC samples benefit from sucrose gradient purification to remove desmoplastic stroma debris. #### Troubleshooting Problem Possible Cause Solution Very low RNA quality High endogenous RNase activity Increase RNase inhibitor to 1.0 U/uL; add VRC; switch to citric acid buffer Under-representation of islet cells Islets are only 1--2% of tissue mass Use NKX2-2-based FACS sorting to enrich endocrine populations High debris from exocrine cells Zymogen granule release Sucrose gradient purification; additional wash cycles Low yield from tumor samples Dense desmoplastic stroma Use Extended protocol on Singulator; increase starting tissue mass #### Singulator Protocol Singulator 100 / 200 / 200+ -- Extended Nuclei Isolation Protocol (recommended for pancreas) Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (1.0 U/uL -- maximum concentration due to pancreatic RNase activity) Sample size : 26--200 mg frozen pancreatic tissue Auto-Mince : Yes Incubation : 5 min at Cold (Extended protocol provides 5 min incubation in NIR) Mixing : Top at Fastest speed Disruption : Default at Fastest speed, with second disruption cycle (Extended protocol) Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; optional sucrose gradient purification for PDAC; filter through 40 um strainer Run time : ~12 min on instrument (Extended protocol) Published use : MSKCC used Singulator 100 with Extended Nuclei Protocol for PDAC snDNA-seq via Mission Bio Tapestri, published in Nature Communications FFPE pancreas : The Singulator 200+ has been validated for FFPE PDAC tissue with the FFPE Nuclei Isolation protocol, demonstrating superior preservation of cancer cell and cancer-associated fibroblast populations compared to manual methods (Precision Cell Systems) #### References [1] Zhang H, Karnoub ER, Umeda S, et al. Application of high-throughput single-nucleus DNA sequencing in pancreatic cancer. Nature Communications. 2023;14:749. [https://www.nature.com/articles/s41467-023-36344-z](https://www.nature.com/articles/s41467-023-36344-z) [2] Park SY, et al. NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing. BMC Genomics. 2024;25:427. [https://link.springer.com/article/10.1186/s12864-024-10335-w](https://link.springer.com/article/10.1186/s12864-024-10335-w) [3] Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) ### Peripheral Nerve #### Overview Peripheral nerve tissue consists of axons wrapped in myelin sheaths (Schwann cells), organized into fascicles by connective tissue (endoneurium, perineurium, epineurium). The tissue is small and fibrous. Nuclei isolation captures Schwann cell nuclei, fibroblast nuclei, and endothelial cell nuclei; neuronal cell bodies are in ganglia, not in the nerve itself. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 15--20 per pestle - Singulator protocol : Extended Nuclei Isolation V2 - Cartridge : NIC+ (nerve segments are typically small) - Expected yield : Low (5,000--20,000 nuclei/mg due to low cellularity) - Key cell types : Schwann cells, fibroblasts (endoneurial, perineurial), endothelial cells - Tips : Myelin debris may be present; consider Nuclei Debris Removal Reagent (Precision Cell Systems) if downstream sequencing quality is affected ### Placenta #### Overview Placenta is a highly vascular, moderately fibrous tissue composed of trophoblast cells (cytotrophoblasts, syncytiotrophoblasts), decidual stromal cells, endothelial cells, Hofbauer cells (fetal macrophages), and maternal immune cells. The tissue has a spongy consistency due to the villous tree structure. Placenta can be collected fresh at delivery, making it one of the few human tissues routinely available in large quantities. Frozen placenta sections are commonly used for nuclei isolation. The Singulator has been used for placenta tissue processing. Enzyme selection can follow the spleen enzyme kit (similar collagenase activity) or a modified brain protocol with 20--30 min incubation for cell isolation (Precision Cell Systems). For nuclei isolation, Standard protocol parameters are appropriate. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen placental tissue. Remove decidua/maternal surface if targeting fetal cells. Weigh 50--100 mg. - Lysis : Add 1 mL cold lysis buffer. Transfer to Dounce homogenizer. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. Placenta disrupts moderately. - Washing : Centrifuge 500g, 5 min, 4 degrees C. RBC lysis with ACK buffer if pellet is red (placenta is blood-rich). - Repeat wash : Resuspend. Centrifuge. Repeat. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining. #### Expected Results - Nuclei yield : Moderate to high; 30,000--150,000 nuclei per mg - Cell type composition : Trophoblast cells, decidual stromal cells, endothelial cells, Hofbauer cells, maternal immune cells (depending on tissue layer sampled) - Downstream compatibility : 10x Chromium, 10x Multiome #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 50--100 mg frozen placental tissue Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : ACK lysis for RBC removal; filter through 40 um strainer Run time : ~7 min on instrument ### Prostate #### Overview Prostate tissue is a glandular organ with moderate fibromuscular stroma. The epithelial compartment contains luminal cells, basal cells, and neuroendocrine cells, while the stroma includes smooth muscle cells, fibroblasts, and immune cells. Prostate cancer research is a major application for snRNA-seq, particularly for profiling tumor microenvironments and clonal heterogeneity. The Singulator has been used for prostate tumor nuclei isolation, with published use for an RPM (Rapid Prostate Model) mouse tumor using the Low Volume Nuclei Isolation protocol. The sample was flash frozen, sliced into ~5 mm x 5 mm pieces, and processed on the Singulator 100. The MSKCC group has also validated Singulator nuclei isolation for prostate tumors. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, slice frozen prostate tissue into ~5 mm pieces. Weigh 20--100 mg. - Lysis : Add 1 mL cold lysis buffer. Transfer to Dounce homogenizer. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend. Repeat twice. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining. #### Expected Results - Nuclei yield : Moderate; 20,000--80,000 nuclei per mg depending on tissue composition - Cell type composition : Luminal epithelial cells, basal cells, neuroendocrine cells, stromal fibroblasts, smooth muscle, endothelial cells, immune cells - Downstream compatibility : 10x Chromium, 10x Multiome, Mission Bio Tapestri (snDNA-seq) #### Singulator Protocol Singulator 100 / 200 / 200+ -- Low Volume or Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 20--100 mg flash-frozen prostate tissue Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Run time : ~7 min on instrument Published use : Mouse RPM tumor processed on Singulator 100 with Low Volume Nuclei Isolation; MSKCC validated for prostate tumors #### References [1] Romero R, Chu T, González-Robles TJ, et al. The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1. Nature Cancer. 2024;5:1641-1659. DOI: 10.1038/s43018-024-00838-6 [2] Masilionis I, Chaudhary O, Chaligne R, Mazutis L. Nuclei extraction for single-cell RNAseq from frozen tissue using Singulator 100. protocols.io. 2022. [https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1](https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1) ### Retina #### Overview Retina is a delicate neural tissue organized into distinct layers (photoreceptor, outer nuclear, inner nuclear, ganglion cell layers). It is highly sensitive to mechanical damage, and the photoreceptors (rods and cones) are particularly fragile. Nuclei isolation is preferred over cell isolation for retina because enzymatic dissociation damages the long outer segments of photoreceptors and disrupts the layered architecture. The tissue is typically very small (mouse retina is approximately 1--3 mg), requiring low-input protocols. The Singulator team has recommended the Extended Nuclei Isolation protocol for retina based on published manual methods, with the option to fall back to the Reverse Nuclei Isolation protocol (single disruption, 5 min incubation, no second disruption) if nuclei appear damaged (Precision Cell Systems). #### Recommended Lysis Buffer Gentle NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis detergent BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, handle retina with extreme care. Mouse retina is very small (1--3 mg); pool multiple retinas if possible. For larger retinas (primate, human), quarter the tissue. - Lysis : Add 0.5--1 mL cold lysis buffer (Low Volume for small samples). Transfer to Dounce homogenizer. - Mechanical disruption : Pestle A, 5--10 gentle strokes. Pestle B, 5--10 gentle strokes. Retina is soft and disrupts easily. - Incubation : 5 min on ice. - Washing : Centrifuge 600g, 5 min, 4 degrees C. Resuspend. - Filtration : Filter through 70 um Flowmi, then 40 um Flowmi. - Quality check : DAPI staining. Target 750--1,200 nuclei/uL for 10x loading. #### Expected Results - Nuclei yield : Moderate; retina is moderately cellular with distinct neuronal layers - Cell type composition : Rod photoreceptors (dominant), cone photoreceptors, bipolar cells, amacrine cells, horizontal cells, ganglion cells, Muller glia, astrocytes, microglia, endothelial cells - Quality considerations : snRNA-seq preserves retinal cell type proportions better than scRNA-seq, which tends to lose photoreceptors - Downstream compatibility : 10x Chromium, 10x Multiome ATAC+GEX, snATAC-seq #### Tips and Tricks - Extended protocol first, Reverse protocol as fallback : Start with Extended Nuclei Isolation V2. If nuclei look damaged, switch to Reverse Nuclei Isolation V2 (single disruption with 5 min incubation, no second disruption) (Precision Cell Systems). - NIC+ cartridge essential : Mouse retina is well below 20 mg. NIC+ cartridge is required for small inputs. - Pool retinas : Multiple mouse retinas (2--4) can be pooled to improve yield and reduce technical variability. - Gentle processing : Retinal neurons are fragile. Use the lowest effective disruption intensity. #### Singulator Protocol Singulator 100 / 200 / 200+ -- Extended Nuclei Isolation Protocol (start here; Reverse protocol as fallback) Cartridge : NIC+ Nuclei Isolation Cartridge (essential for small retina samples) Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 1--10 mg (mouse retina); 20--50 mg (human/primate retina) Auto-Mince : Yes Incubation : 5 min at Cold (Extended) Mixing : Top at Medium speed (gentle for neural tissue) Disruption : Default at Medium speed, dual disruption cycle (Extended); or single disruption (Reverse) if nuclei appear damaged Run time : ~12 min on instrument (Extended) Recommendation : "I would recommend the Extended Nuclei Isolation protocol. If the nuclei look damaged, my next step would be the Reverse Nuclei Isolation protocol." (Precision Cell Systems) #### References [1] Protocols.io. Nuclei Isolation for SnRNA-seq and SnATAC-seq from Frozen Fresh Human Retina Sample. [https://www.protocols.io/view/nuclei-isolation-for-snrna-seq-and-snatac-seq-from-bftmjnk6](https://www.protocols.io/view/nuclei-isolation-for-snrna-seq-and-snatac-seq-from-bftmjnk6) [2] Lukowski SW, et al. Single-nuclei RNA-seq on human retinal tissue provides improved transcriptome profiling. Nature Communications. 2019;10:5743. [https://www.nature.com/articles/s41467-019-12917-9](https://www.nature.com/articles/s41467-019-12917-9) ### Salivary Gland #### Overview Salivary glands are exocrine glands composed of acinar cells (serous and mucous), ductal cells, myoepithelial cells, and a connective tissue stroma. The tissue is moderately soft and amenable to standard nuclei isolation. Like pancreas, salivary gland has elevated RNase activity from enzymatic secretions, though less extreme. A validated Singulator protocol exists for mouse salivary gland nuclei isolation (10--25 mg, Standard Nuclei Isolation, Fastest mixing speed, Medium disruption speed, with FACS sorting). #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 10--15 per pestle - Singulator protocol : Standard Nuclei Isolation V2 - Cartridge : NIC+ (salivary glands are often 10--25 mg in mice) - Expected yield : Moderate (20,000--60,000 nuclei/mg) - Key cell types : Acinar cells (serous/mucous), ductal cells, myoepithelial cells, stromal cells - Tips : Medium disruption speed on Singulator; FACS sorting recommended; clean blade with RNAzap before cutting tissue (Precision Cell Systems) #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (700 units per run) Sample size : 10--25 mg frozen salivary gland (cut half gland with RNAzap-cleaned blade on dry ice) (Precision Cell Systems) Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Medium speed (Precision Cell Systems) Post-processing : Transfer to 5 mL glass FACS tube; centrifuge 600g, 8 min, 4 degrees C; resuspend; FACS sort for single nuclei (Precision Cell Systems) Run time : ~7 min on instrument ### Skin #### Overview Skin is one of the most challenging tissues for nuclei isolation due to its dense extracellular matrix, high collagen content, and elastic mechanical properties. The epidermis contains tightly packed keratinocytes connected by desmosomes and tight junctions, while the dermis is composed of dense connective tissue rich in collagen I and III, elastin, and glycosaminoglycans. Hair follicles add an additional structural element. These properties make skin tissue resistant to mechanical disruption, and nuclei yields tend to be lower compared to softer tissues. Cell isolation from fresh skin tissue has been validated on the Singulator, yielding approximately 5,200 cells per mg at 90% viability with identification of all major cell types including permanent epidermal keratinocytes, fibroblasts, anagen hair follicle keratinocytes, T-cells, endothelial cells, Schwann cells, and melanocytes. For nuclei isolation from frozen skin, the Extended Nuclei Isolation protocol is recommended as the starting point because of the tissue's elastic, difficult-to-disrupt nature. #### Recommended Lysis Buffer NP-40 lysis buffer with higher detergent : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1--0.2% May need higher concentration due to collagen resistance Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : Trim fur (if present). On dry ice, mince frozen skin tissue into the finest possible pieces (sub-millimeter). Skin is elastic and resists mincing; use a sharp, cold blade. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 20--25 strokes (skin requires extra force). Pestle B, 20--25 strokes. Tissue may not fully homogenize; residual fragments are normal. - Extended incubation : 5--10 min on ice in lysis buffer to complete nuclear release from collagen-rich matrix. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat twice. - Filtration : Sequential filtration through 70 um then 40 um strainers. - Quality check : DAPI staining. Expect lower yield per mg compared to brain or kidney. #### Expected Results - Nuclei yield : Low; approximately 5,000--15,000 nuclei per mg depending on tissue preparation and disruption intensity - Cell type composition : Keratinocyte nuclei predominate, along with fibroblasts, endothelial cells, immune cells, melanocytes, Schwann cells - Quality metrics : 2,302 median genes per cell; 20,092 mean reads per cell; 88% of cells with #### Tips and Tricks - Extended protocol is typically needed : Standard nuclei isolation is generally insufficient for skin. The Extended protocol with 5 min incubation and second disruption cycle is the recommended starting point. - Fine mincing is important : Finer mincing before loading generally improves nuclei yield. Use scissors on dry ice to achieve the finest possible fragments. - Oral mucosa is similar : Buccal mucosa and tongue epithelium share characteristics with skin (elastic, keratin-rich). The same Extended protocol applies; validated protocols for mouse buccal mucosa use the Low Volume 2x Disruption protocol with NIC+ cartridge. - Decrease speed if nuclei are damaged : Start with Extended at Fastest speed but reduce disruption speed if nuclei appear damaged under microscopy. - Frozen preferred for nuclei : Frozen skin tissue disrupts more readily than fresh due to ice crystal formation breaking cell membranes. #### Troubleshooting Problem Possible Cause Solution Very low yield Elastic tissue resists disruption Mince more finely; use Extended or 2x Disruption protocol; increase starting material Residual tissue fragments Dense collagen matrix Normal for skin; ensure fragments do not clog filters; try additional disruption cycle Keratinocyte dominance Expected tissue composition Computational gating for cell type annotation Debris from hair follicles Follicular structures Additional filtration through 20 um strainer #### Singulator Protocol Singulator 100 / 200 / 200+ -- Extended Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 20--100 mg frozen skin tissue Auto-Mince : Yes Incubation : 5 min at Cold (Extended protocol with 5 min incubation in NIR) Mixing : Top at Fastest speed Disruption : Default at Fastest speed, with second disruption cycle Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; filter through 40 um strainer Run time : ~12 min on instrument (Extended protocol) Guidance : "Nuclei is tough with skin. I would recommend the Extended Nuclei Isolation as a good starting point. Since the tissue is elastic in nature, it is harder to dissociate and isolate nuclei. Mincing the tissue before as well will help increase the yield." (Precision Cell Systems) #### References [1] Precision Cell Systems. Application Note: Isolation of Mouse Skin Cells. Singulator 100 cell isolation yielding 5,200 cells/mg at 90% viability with scRNA-seq via 10x Genomics Chromium. ### Spinal Cord #### Overview Spinal cord is a neural tissue similar to brain in composition, containing neurons, astrocytes, oligodendrocytes, and microglia, but with a higher proportion of white matter (myelinated axons). Myelin debris is a major contaminant, as with brain tissue. Standard brain nuclei isolation protocols apply, with mandatory myelin/debris removal. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) -- same as brain - Dounce strokes : 10--15 per pestle - Singulator protocol : Low Volume Nuclei Isolation V2 (same as brain) - Cartridge : NIC+ with Nuclei Debris Removal Reagent for myelin removal - Expected yield : Similar to brain (30,000--100,000 nuclei/mg); lower than cerebellum but comparable to cortex - Key cell types : Motor neurons, interneurons, astrocytes, oligodendrocytes, microglia, ependymal cells - Tips : Treat identically to brain for nuclei isolation; myelin debris removal is generally necessary; RNase inhibitor at 0.2 U/uL is still recommended (neural tissue has lower RNase activity than many other tissues) ### Spleen #### Overview Spleen is a highly cellular lymphoid organ with two main compartments: the red pulp (filtering blood, removing senescent red blood cells) and the white pulp (containing organized lymphoid tissue for immune surveillance). Spleen is relatively soft and easy to disrupt mechanically, producing high nuclei yields. However, two significant challenges must be managed: (1) heavy red blood cell contamination from the blood-filtering function of the red pulp, and (2) high endogenous RNase activity, requiring elevated RNase inhibitor concentrations. Spleen nuclei isolation has been validated on the Singulator using the Standard Nuclei Isolation protocol, with published use in multi-species studies including cynomolgus macaque spleen. The tissue is well-suited for the Singulator platform because of its soft consistency and high cellularity. #### Recommended Lysis Buffer Standard NP-40 lysis buffer with elevated RNase protection : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4--1.0 U/uL Higher concentration needed due to high RNase expression #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen spleen tissue. Spleen is soft and minces easily. Weigh 50--60 mg. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. Spleen disrupts easily; avoid over-processing. - Washing : Centrifuge 500g, 5 min, 4 degrees C. - RBC lysis : Resuspend pellet in 1 mL ACK lysis buffer (150 mM NH4Cl, 10 mM KHCO3, 0.1 mM EDTA, pH 7.2--7.4). Incubate 3 min on ice. Centrifuge 500g, 5 min, 4 degrees C. - Additional wash : Resuspend in wash buffer with RNase inhibitor. Centrifuge. Repeat once. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining. Verify minimal RBC contamination. #### Expected Results - Nuclei yield : High; spleen is among the most cellular tissues, yielding 50,000--200,000 nuclei per mg - Cell type composition : T cells, B cells, macrophages, dendritic cells, NK cells, granulocytes, stromal cells, red pulp cells - Processing time : 25 min on Singulator (including instrument time plus wash/RBC lysis) - Downstream compatibility : 10x Chromium, BD Rhapsody, 10x Immune Profiling (5' VDJ) #### Tips and Tricks - RBC lysis is strongly recommended : Spleen typically contains large amounts of blood. Including an ACK lysis step after initial centrifugation is standard practice. - Higher RNase inhibitor : Spleen has high endogenous RNase expression. Use 0.4--1.0 U/uL RNase inhibitor in all buffers. - Process promptly : High RNase activity means spleen nuclei are more susceptible to RNA degradation during extended hold times. Process downstream within 30 min of isolation. - Standard protocol is sufficient : Spleen is soft; the Standard Nuclei Isolation protocol with default settings works well. No need for Extended protocol. - Multi-species validated : The Singulator Low Volume Nuclei Isolation protocol has been published for cynomolgus macaque spleen tissue. #### Troubleshooting Problem Possible Cause Solution Red pellet RBC contamination Add ACK lysis step (3 min on ice); repeat if pellet is still red RNA degradation High RNase activity Increase RNase inhibitor to 1.0 U/uL; add RNase inhibitor to all buffers including ACK Immune cell bias Expected tissue composition Normal for spleen; all major immune populations should be recovered Over-disruption Spleen is very soft Reduce Dounce strokes (8--10 per pestle); lower Singulator speed #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4--1.0 U/uL; use higher concentration for spleen due to RNase expression) Sample size : 50--60 mg frozen spleen tissue Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; ACK lysis buffer for 3 min on ice; second centrifugation; resuspend in NSR + RNase inhibitor; filter through 40 um strainer Run time : ~7 min on instrument plus ~15 min wash/RBC lysis Published use : Roche Innovation Centre Basel published Singulator 100 nuclei isolation from cynomolgus macaque spleen in JOVE (2023) #### References [1] Nobs SP, et al. A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing. JOVE. 2023;(197):e65611. [https://www.jove.com/t/65611/a-simple-quick-partially-automated-protocol-for-isolation-single](https://www.jove.com/t/65611/a-simple-quick-partially-automated-protocol-for-isolation-single) ### Stomach #### Overview Stomach tissue has a glandular mucosa (containing parietal cells, chief cells, mucous cells, enteroendocrine cells, and stem cells), submucosa, muscularis propria, and serosa. The mucous layer can interfere with nuclei isolation. The tissue is moderately soft; Standard to Extended protocols are appropriate. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 10--15 per pestle - Singulator protocol : Standard or Extended Nuclei Isolation V2 - Expected yield : Moderate (20,000--80,000 nuclei/mg) - Key cell types : Parietal cells, chief cells, mucous cells, enteroendocrine cells, stem cells, smooth muscle, immune cells - Tips : Remove luminal contents; wash tissue in cold PBS before freezing if possible; process similarly to intestine ### Testis #### Overview Testis tissue contains the seminiferous tubules (housing spermatogonia, spermatocytes, spermatids at various stages, and Sertoli cells) and the interstitial compartment (Leydig cells, macrophages, endothelial cells). The tissue is moderately soft and can be disrupted with standard mechanical methods. A unique consideration is the presence of highly condensed spermatid nuclei, which are transcriptionally silent and should be excluded from snRNA-seq analysis computationally. The Singulator has been validated for testis tissue nuclei isolation. The Standard Nuclei Isolation protocol is appropriate for this moderately soft tissue. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent BSA 1% Carrier protein RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen testis tissue. Remove tunica albuginea (capsule) if accessible. Weigh 30--60 mg. - Lysis : Add 1 mL cold lysis buffer. Transfer to Dounce homogenizer. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend. Repeat. - Filtration : Filter through 40 um strainer. Consider additional 20 um filtration to remove spermatozoa. - Quality check : DAPI staining. Spermatids will be abundant and have very small, condensed nuclei. #### Expected Results - Nuclei yield : Moderate to high; 30,000--100,000 nuclei per mg - Cell type composition : Spermatogonia, spermatocytes, round spermatids, elongating spermatids, Sertoli cells, Leydig cells, macrophages, endothelial cells - Downstream compatibility : 10x Chromium, 10x Multiome #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 30--60 mg frozen testis tissue Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Run time : ~7 min on instrument ### Thymus #### Overview Thymus is a primary lymphoid organ responsible for T cell maturation. It has a cortex (densely packed with immature thymocytes) and a medulla (more loosely organized with mature thymocytes and thymic epithelial cells). Like other lymphoid organs, thymus is highly cellular and relatively soft, making nuclei isolation straightforward. A validated Singulator protocol exists for chicken thymus nuclei isolation (50--60 mg, Standard Nuclei Isolation, Fastest speed, with 40 um filtration). USDA researchers have also used the Singulator for pig thymus nuclei isolation. #### Recommended Lysis Buffer Standard NP-40 lysis buffer : Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Standard lysis BSA 1% Carrier protein RNase inhibitor 0.4 U/uL RNA preservation #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen thymus tissue. Weigh 50--60 mg. - Lysis : Add 1 mL cold lysis buffer. Transfer to Dounce homogenizer. - Mechanical disruption : Pestle A, 10--15 strokes. Pestle B, 10--15 strokes. Thymus is soft and disrupts easily. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend. Repeat. - Filtration : Filter through 40 um strainer. - Quality check : DAPI staining. #### Expected Results - Nuclei yield : High; thymus is densely cellular (50,000--200,000 nuclei per mg) - Cell type composition : Thymocytes (double-positive, single-positive CD4, single-positive CD8), thymic epithelial cells (cortical and medullary), dendritic cells, macrophages - Downstream compatibility : 10x Chromium, 10x Immune Profiling, BD Rhapsody, CITE-seq #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge (or standard blue for 50+ mg) Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL) Sample size : 50--60 mg frozen thymus tissue Pre-cooling : Cool Singulator for approximately 15 min before loading Auto-Mince : Yes Incubation : 0 min at Cold Mixing : Top at Fastest speed Disruption : Default at Fastest speed Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in 1,000 uL NSR + 0.4 U/uL RNase inhibitor; second centrifugation; final resuspension in 500 uL NSR + 0.4 U/uL RNase inhibitor; filter through 40 um strainer (Precision Cell Systems) Run time : ~7 min on instrument ### Thyroid #### Overview Thyroid tissue consists of follicles lined by follicular epithelial cells (thyrocytes) surrounding colloid-filled lumina, interspersed with parafollicular C cells. The tissue is soft, moderately cellular, and amenable to standard nuclei isolation protocols. The colloid (stored thyroglobulin) may create debris that should be removed by washing. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%) - Dounce strokes : 10--15 per pestle - Singulator protocol : Standard Nuclei Isolation V2 - Cartridge : NIC+ (thyroid glands are typically small) - Expected yield : Moderate (20,000--60,000 nuclei/mg) - Key cell types : Follicular epithelial cells (thyrocytes), parafollicular C cells, endothelial cells, fibroblasts - Tips : Multiple washes to remove colloid; standard processing parameters ### Tumors (Solid) #### Overview Solid tumors represent one of the most heterogeneous and variable tissue types for nuclei isolation. Each tumor type has distinct properties: gliomas are soft and lipid-rich, pancreatic tumors are surrounded by dense desmoplastic stroma, breast tumors range from soft to fibrotic, and melanomas vary based on anatomical location. Despite this variability, nuclei isolation from frozen solid tumors is well-established and frequently preferred over cell isolation because it enables profiling of biobanked specimens, eliminates dissociation-induced transcriptional artifacts, and captures fragile cell types that are lost during enzymatic digestion. The Singulator has been extensively validated for tumor nuclei isolation, with published use in glioblastoma (UCSF), pancreatic ductal adenocarcinoma (MSKCC), prostate tumors, breast tumors, melanoma, colon tumors, and nasopharyngeal tumors. The Extended Nuclei Isolation protocol is recommended for tumors that appear under-dissociated, while Standard or Low Volume protocols work well for softer tumor types. #### Recommended Lysis Buffer Standard NP-40 lysis buffer (suitable for most tumor types): Component Concentration Notes Tris-HCl pH 7.4 10 mM Base buffer NaCl 10 mM Osmotic balance MgCl2 3 mM Nuclear integrity NP-40 / IGEPAL CA-630 0.1% Lysis detergent Tween-20 0.1% Anti-aggregation BSA 1% Carrier protein DTT 1 mM Reducing agent RNase inhibitor 0.4--1.0 U/uL Adjust based on tumor type Alternative for fibrous tumors : IGEPAL-based buffer with 5 min incubation for improved nuclear release from dense stroma. #### Step-by-Step Protocol - Tissue preparation : On dry ice, mince frozen tumor tissue into sub-millimeter pieces. For soft tumors (glioma, melanoma), minimal mincing is needed. For fibrous tumors (breast, PDAC), mince as finely as possible. - Lysis : Add 1 mL cold lysis buffer to Dounce homogenizer. Transfer tissue. - Mechanical disruption : Pestle A, 10--20 strokes (adjust based on tumor firmness). Pestle B, 10--20 strokes. - Incubation (for dense tumors): 5 min on ice in lysis buffer. - Washing : Centrifuge 500g, 5 min, 4 degrees C. Resuspend in wash buffer. Repeat twice. - Debris removal (recommended for tumors with necrotic cores): Percoll gradient or density-based cleanup. - Filtration : Filter through 40 um strainer. Sequential filtration through 30 um MARC SmartStrainer (2x) and 40 um Flowmi strainer has been published for brain tumor nuclei. - Quality check : DAPI staining and counting. Inspect for necrotic debris. #### Expected Results - Nuclei yield : Highly variable (8,000--500,000 nuclei/mg) depending on tumor type, cellularity, and necrosis - Cell type composition : Tumor cells, stromal cells (fibroblasts, cancer-associated fibroblasts), endothelial cells, immune infiltrate (T cells, macrophages, neutrophils, NK cells) - Quality metrics : Human glioblastoma nuclei isolated with Singulator 100 yielded sufficient quality for 10x Chromium snRNA-seq and snATAC-seq - Downstream compatibility : 10x Chromium, Mission Bio Tapestri (snDNA-seq), 10x Multiome, 10x Flex Assay #### Tips and Tricks - Start with Standard or Low Volume; escalate to Extended : Begin with Standard Nuclei Isolation V2 for soft tumors (glioma, melanoma). If tissue appears under-dissociated, switch to Extended Nuclei Isolation V2. - Tumor heterogeneity requires replicate processing : Different regions of the same tumor may yield dramatically different results. Process multiple regions when possible. - FACS sorting improves purity : For tumors with high necrosis, sorting DAPI-positive nuclei removes necrotic debris and dramatically improves sequencing quality. - FFPE tumors on Singulator 200+ : For archived FFPE tumor samples, the Singulator 200+ with its two-step FFPE workflow (deparaffinization cartridge followed by NIC+ cartridge) provides automated nuclei isolation with 81% reduction in hands-on time compared to manual methods (Precision Cell Systems). - Reduce sample mass for Low Volume protocol : Brain tumors at 130 mg were too large for Low Volume protocol; 75 mg processed fully on NIC+ cartridge (Precision Cell Systems). #### Troubleshooting Problem Possible Cause Solution Under-dissociated tissue Dense stroma (PDAC, breast) Use Extended protocol with 5 min incubation and second disruption; mince more finely High necrotic debris Tumor core necrosis Percoll gradient cleanup; FACS sort DAPI+ nuclei; avoid visibly necrotic regions Low tumor cell recovery Immune infiltrate dominance Expected in immune-hot tumors; computational deconvolution identifies cell populations Variable yield across replicates Tumor heterogeneity Process multiple regions; standardize mincing and sample mass #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard or Extended Nuclei Isolation Protocol (choose based on tumor type) Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL standard; 1.0 U/uL for RNase-rich tumors) Sample size : 20--100 mg frozen tumor tissue (75 mg optimal for brain tumors; up to 200 mg for PDAC) Auto-Mince : Yes Incubation : 0 min (Standard) or 5 min at Cold (Extended) -- use Extended for dense/fibrous tumors Mixing : Top at Fastest speed Disruption : Default at Fastest speed (Standard) or dual disruption cycle (Extended) Post-processing : Centrifuge 500g, 5 min, 4 degrees C; resuspend in NSR + RNase inhibitor; optional density-based debris removal; filter through 40 um strainer; optional FACS sorting Run time : ~7 min (Standard) or ~12 min (Extended) on instrument Published tumor types on Singulator : Glioblastoma (UCSF), PDAC (MSKCC, Nature Communications), prostate tumors, melanoma, colon tumors (MSKCC, Science Translational Medicine), nasopharyngeal tumors, brain tumors with melanoma metastasis (MSKCC, Singulator 200+ FFPE) (Precision Cell Systems) FFPE tumors : Singulator 200+ exclusive -- two-step cartridge workflow (FFPE cartridge for deparaffinization + NIC+ cartridge for nuclei isolation); validated on FFPE PDAC with superior cancer cell recovery compared to manual methods (Precision Cell Systems) #### References [1] Slyper M, Porter CBM, Ashenberg O, et al. A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors. Nature Medicine. 2020;26:792-802. [https://www.nature.com/articles/s41591-020-0844-1](https://www.nature.com/articles/s41591-020-0844-1) [2] Masilionis I, Chaudhary O, Chaligne R, Mazutis L. Nuclei extraction for single-cell RNAseq from frozen tissue using Singulator 100. protocols.io. 2022. [https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1](https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1) [3] Zhang H, Karnoub ER, Umeda S, et al. Application of high-throughput single-nucleus DNA sequencing in pancreatic cancer. Nature Communications. 2023;14:749. [https://www.nature.com/articles/s41467-023-36344-z](https://www.nature.com/articles/s41467-023-36344-z) [4] Romero R, Chu T, González-Robles TJ, et al. The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1. Nature Cancer. 2024;5:1641-1659. DOI: 10.1038/s43018-024-00838-6 [5] Haegebarth A, et al. Improved protocol for single-nucleus RNA-sequencing of frozen human bladder tumor biopsies. Nucleus. 2023;14(1):2186686. [https://pmc.ncbi.nlm.nih.gov/articles/PMC10012951/](https://pmc.ncbi.nlm.nih.gov/articles/PMC10012951/) [6] Strobl J, et al. Tissue-specific features of the T cell repertoire after allogeneic hematopoietic cell transplantation in human and mouse. Science Translational Medicine. 2023;15:eabq0476. [https://www.science.org/doi/10.1126/scitranslmed.abq0476](https://www.science.org/doi/10.1126/scitranslmed.abq0476) ### Zebrafish #### Overview Zebrafish (Danio rerio) are a widely used vertebrate model organism for developmental biology, genetics, and disease modeling. Key tissues processed for nuclei isolation include fins (regeneration studies), embryos (developmental biology), brain, and internal organs. Zebrafish tissues are generally soft and small, but have unique considerations: (1) scales and fin rays provide some mechanical resistance, (2) ambient temperature is 28--30 degrees C rather than 37 degrees C, and (3) zebrafish genomes have undergone a whole-genome duplication, increasing the complexity of downstream analysis. The Singulator has been used for zebrafish fin tissue and embryo cell isolation at the University of Oregon, using enzymatic digestion protocols with liberase and accutase. For nuclei isolation from frozen zebrafish tissue, standard mammalian protocols can be adapted with minor modifications. #### Recommended Protocol - Lysis buffer : Standard NP-40 (0.1%), 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 0.4 U/uL RNase inhibitor - Dounce strokes : 10--15 per pestle for soft tissues (brain, viscera); 15--20 for fins - Singulator protocol : Standard or Low Volume Nuclei Isolation V2 (Low Volume for small embryo/tissue samples) - Cartridge : NIC+ (zebrafish tissues are often small) - Expected yield : Variable by tissue; brain and viscera yield moderate nuclei; fins yield fewer - Key cell types : Tissue-dependent; fins contain osteoblasts, fibroblasts, melanocytes, epidermis; embryos contain diverse progenitor populations - Tips : Zebrafish tissues are small -- pool multiple animals (20+ fins) for adequate yield; for embryos, enzymatic pre-digestion may improve nuclear release from yolk-containing stages; NIC+ essential for small samples #### Singulator Protocol Singulator 100 / 200 / 200+ -- Standard or Low Volume Nuclei Isolation Protocol Cartridge : NIC+ Nuclei Isolation Cartridge Reagents : NSR + NIR + RNase inhibitor (0.4 U/uL); or use Single-Shot with custom enzymes (e.g., liberase, trypsin-EDTA, accutase) for cell isolation Sample size : Variable; pool 20+ fins or multiple embryos for adequate mass Published use : University of Oregon used Singulator for zebrafish fin tissue with enzymatic digestion protocols ## Bibliography & Source Index Protocols, publications, and application notes referenced across the tissue sections above. Cell isolation protocols include per-section reference footnotes. - protocols.io: Brain Tissue Dissociation for Cell Sorting. Grant Lin, Michelle Monje (Stanford). [https://www.protocols.io/view/brain-tissue-dissociation-for-cell-sorting-nx7dfrn](https://www.protocols.io/view/brain-tissue-dissociation-for-cell-sorting-nx7dfrn) - protocols.io: Dissociation of Single Cell Suspensions from Human Breast Tissues. Nicholas Navin (MD Anderson). [https://www.protocols.io/view/dissociation-of-single-cell-suspensions-from-human-t3geqjw](https://www.protocols.io/view/dissociation-of-single-cell-suspensions-from-human-t3geqjw) - protocols.io: Breast tumours dissociation. Princess Margaret Cancer Centre. [https://www.protocols.io/view/breast-tumours-dissociation-7m9hk96](https://www.protocols.io/view/breast-tumours-dissociation-7m9hk96) - protocols.io: Human breast tissue dissociation and FACS vs Flowmi for scRNA-Seq. UC Irvine. [https://www.protocols.io/view/human-breast-tissue-dissociation-and-facs-vs-flowm-bu2qnydw](https://www.protocols.io/view/human-breast-tissue-dissociation-and-facs-vs-flowm-bu2qnydw) - protocols.io: Human colon tissue dissociation for immune cells. Kylie James (Sanger). [https://www.protocols.io/view/human-colon-tissue-dissociation-for-immune-cells-tbfeijn](https://www.protocols.io/view/human-colon-tissue-dissociation-for-immune-cells-tbfeijn) - protocols.io: Epithelial/Immune Dissociation for Human Colon Biopsies. Broad Institute. [https://www.protocols.io/view/epithelial-immune-dissociation-for-human-colon-bio-32igqce](https://www.protocols.io/view/epithelial-immune-dissociation-for-human-colon-bio-32igqce) - protocols.io: Dissociation of fresh colorectal biopsies. Vanderbilt. [https://www.protocols.io/view/dissociation-of-fresh-colorectal-biopsies-bci4iugw](https://www.protocols.io/view/dissociation-of-fresh-colorectal-biopsies-bci4iugw) - protocols.io: Dissociation of Jejunum cells for clumps sorting. Weizmann Institute. [https://www.protocols.io/view/dissociation-of-jejunum-cells-for-clumps-sorting-bvq3n5yn](https://www.protocols.io/view/dissociation-of-jejunum-cells-for-clumps-sorting-bvq3n5yn) - protocols.io: JAX-Sen: Mouse heart dissociation for scRNA-seq. Jackson Lab. [https://www.protocols.io/view/jax-sen-mouse-heart-dissociation-for-single-cell-r-dgkw3uxe](https://www.protocols.io/view/jax-sen-mouse-heart-dissociation-for-single-cell-r-dgkw3uxe) - protocols.io: Adult human kidney tissue cell dissociation (on ice). Andrew Potter (U Cincinnati). [https://www.protocols.io/view/adult-human-kidney-tissue-cell-dissociation-on-ice-q6cdzaw](https://www.protocols.io/view/adult-human-kidney-tissue-cell-dissociation-on-ice-q6cdzaw) - protocols.io: JAX-Sen: Mouse kidney dissociation for scRNA-seq. Jackson Lab. [https://www.protocols.io/view/jax-sen-mouse-kidney-dissociation-for-single-cell-dgkv3uw6](https://www.protocols.io/view/jax-sen-mouse-kidney-dissociation-for-single-cell-dgkv3uw6) - protocols.io: Human Kidney Tumor Dissociation for single-cell genomics. Univ Rennes/Inserm. [https://www.protocols.io/view/human-kidney-tumor-dissociation-for-single-cell-g-2n2gdge](https://www.protocols.io/view/human-kidney-tumor-dissociation-for-single-cell-g-2n2gdge) - protocols.io: Human Liver Tissue Dissociation for 10x Single Cell RNA-seq. Ankur Sharma (GIS). [https://www.protocols.io/view/human-liver-tissue-dissociation-for-10x-single-cel-vhue36w](https://www.protocols.io/view/human-liver-tissue-dissociation-for-10x-single-cel-vhue36w) - protocols.io: Human Liver Caudate Lobe Dissociation for ScRNA-seq v2. Sonya MacParland (U Toronto). [https://www.protocols.io/view/human-liver-caudate-lobe-dissociation-for-scrna-se-m9sc96e](https://www.protocols.io/view/human-liver-caudate-lobe-dissociation-for-scrna-se-m9sc96e) - protocols.io: Adult mouse lung cell dissociation (on ice) v4. Andrew Potter (CCHMC). [https://www.protocols.io/view/adult-mouse-lung-cell-dissociation-on-ice-cfiytkfw](https://www.protocols.io/view/adult-mouse-lung-cell-dissociation-on-ice-cfiytkfw) - protocols.io: Adult human lung cell dissociation (on ice) v2. Andrew Potter (CCHMC). [https://www.protocols.io/view/adult-human-lung-cell-dissociation-on-ice-ch6dt9a6](https://www.protocols.io/view/adult-human-lung-cell-dissociation-on-ice-ch6dt9a6) - protocols.io: Single Cell Dissociation of Fresh Lung Tissue. Lance Peter, Mei-I Chung (TGen). [https://www.protocols.io/view/single-cell-dissociation-of-fresh-lung-tissue-7xkhpkw](https://www.protocols.io/view/single-cell-dissociation-of-fresh-lung-tissue-7xkhpkw) - protocols.io: Cell dissociation of fresh human lung tissue for scRNA-seq. Ilias Angelidis (Helmholtz Munich). [https://www.protocols.io/view/cell-dissociation-of-fresh-human-lung-tissue-for-s-zp2f5qe](https://www.protocols.io/view/cell-dissociation-of-fresh-human-lung-tissue-for-s-zp2f5qe) - protocols.io: Adult mouse pancreas cell dissociation (on ice) v2. Andrew Potter (CCHMC). [https://www.protocols.io/view/adult-mouse-pancreas-cell-dissociation-on-ice-cfi3tkgn](https://www.protocols.io/view/adult-mouse-pancreas-cell-dissociation-on-ice-cfi3tkgn) - protocols.io: Human skin single cell dissociation. Newcastle/Sanger. [https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn](https://www.protocols.io/view/human-skin-single-cell-dissociation-ripd4dn) - protocols.io: Adult Mouse Spleen Dissociation (On ice) v3. Andrew Potter (CCHMC). [https://www.protocols.io/view/adult-mouse-spleen-dissociation-on-ice-ydwfs7e](https://www.protocols.io/view/adult-mouse-spleen-dissociation-on-ice-ydwfs7e) - protocols.io: Single Cell Dissociation of Small Tumor Biopsies. ETH Zurich/CZ Biohub. [https://www.protocols.io/view/single-cell-dissociation-of-small-tumor-biopsies-65rhg56](https://www.protocols.io/view/single-cell-dissociation-of-small-tumor-biopsies-65rhg56) - protocols.io: Miltenyi Biotec: Adipose Tissue Dissociation. U Minnesota. [https://www.protocols.io/view/miltenyi-biotec-adipose-tissue-dissociation-univer-ctpywmpw](https://www.protocols.io/view/miltenyi-biotec-adipose-tissue-dissociation-univer-ctpywmpw) - protocols.io: Endometrium dissociation with collagenase. Sanger Institute. [https://www.protocols.io/view/endometrium-dissociation-with-collagenase-76thren](https://www.protocols.io/view/endometrium-dissociation-with-collagenase-76thren) - protocols.io: Human endometrium and endometriosis tissue dissociation for scRNA-seq. Jackson Lab. [https://www.protocols.io/view/human-endometrium-and-endometriosis-tissue-dissoci-bvy8n7zw](https://www.protocols.io/view/human-endometrium-and-endometriosis-tissue-dissoci-bvy8n7zw) - protocols.io: Myometrium Single Cell Dissociation Protocol. U Michigan. [https://www.protocols.io/view/myometrium-single-cell-dissociation-protocol-bmbek2je](https://www.protocols.io/view/myometrium-single-cell-dissociation-protocol-bmbek2je) - protocols.io: CGAP Human Oesophagus Epithelium Dissociation. CGAP. [https://www.protocols.io/view/cgap-human-oesophagus-epithelium-dissociation-qz8dx9w](https://www.protocols.io/view/cgap-human-oesophagus-epithelium-dissociation-qz8dx9w) - protocols.io: Human Fallopian Tube and Ovary Dissociation for scRNA-seq. U Chicago. [https://www.protocols.io/view/human-fallopian-tube-and-ovary-dissociation-for-si-bfudjns6](https://www.protocols.io/view/human-fallopian-tube-and-ovary-dissociation-for-si-bfudjns6) - protocols.io: JAX-Sen: Single Cell Dissociation of Mouse Placenta. Jackson Lab. [https://www.protocols.io/view/jax-sen-single-cell-dissociation-of-mouse-placenta-hcnzb2vf7](https://www.protocols.io/view/jax-sen-single-cell-dissociation-of-mouse-placenta-hcnzb2vf7) - protocols.io: Single-Cell Dissociation of Human Trabecular Meshwork. Harvard/Mass Eye and Ear. [https://www.protocols.io/view/single-cell-dissociation-of-human-trabecular-meshw-bfdyji7w](https://www.protocols.io/view/single-cell-dissociation-of-human-trabecular-meshw-bfdyji7w) - protocols.io: Adult mouse testis cell dissociation (on ice). CCHMC. [https://www.protocols.io/view/adult-mouse-testis-cell-dissociation-on-ice-smkec4w](https://www.protocols.io/view/adult-mouse-testis-cell-dissociation-on-ice-smkec4w) - protocols.io: Human Thymus single cell dissociation protocol. Teichmann Lab (Sanger). [https://www.protocols.io/view/human-thymus-single-cell-dissociation-protocol-tei-bx8sprwe](https://www.protocols.io/view/human-thymus-single-cell-dissociation-protocol-tei-bx8sprwe) - protocols.io: Adult mouse ear dissociation (on ice). Andrew Potter (CCHMC). [https://www.protocols.io/view/adult-mouse-ear-dissociation-on-ice-ucwesxe](https://www.protocols.io/view/adult-mouse-ear-dissociation-on-ice-ucwesxe) - protocols.io: Ganglia dissociation and single-cell sorting. Columbia. [https://www.protocols.io/view/ganglia-dissociation-and-single-cell-sorting-b62frgbn](https://www.protocols.io/view/ganglia-dissociation-and-single-cell-sorting-b62frgbn) - protocols.io: Zebrafish Embryo Dissociation for MACS v2. Harvard/BCH. [https://www.protocols.io/view/zebrafish-embryo-dissociation-for-macs-bhquj5ww](https://www.protocols.io/view/zebrafish-embryo-dissociation-for-macs-bhquj5ww) - protocols.io: Zebrafish larvae dissociation for FACS sorting. Edinburgh. [https://www.protocols.io/view/zebrafish-larvae-dissociation-for-facs-sorting-cel-bzh8p39w](https://www.protocols.io/view/zebrafish-larvae-dissociation-for-facs-sorting-cel-bzh8p39w) - protocols.io: Single cell dissociation of brain organoids. DZNE Tubingen. [https://www.protocols.io/view/single-cell-dissociation-of-brain-organoids-crwsv7ee](https://www.protocols.io/view/single-cell-dissociation-of-brain-organoids-crwsv7ee) - protocols.io: Dissociation of EBs using Worthington Kit. NYSCF. [https://www.protocols.io/view/dissociation-of-ebs-using-worthington-kit-dftv3nn6](https://www.protocols.io/view/dissociation-of-ebs-using-worthington-kit-dftv3nn6) - protocols.io: Tissue Dissociation for Multiome Analysis Using S2 Singulator. UPenn. [https://www.protocols.io/view/tissue-dissociation-for-multiome-analysis-using-s2-ce9cth2w](https://www.protocols.io/view/tissue-dissociation-for-multiome-analysis-using-s2-ce9cth2w) - protocols.io: Manual Tissue Dissociation for Multiome Analysis. UPenn. [https://www.protocols.io/view/manual-tissue-dissociation-for-multiome-analysis-cev5te86](https://www.protocols.io/view/manual-tissue-dissociation-for-multiome-analysis-cev5te86) - Masilionis I, Chaudhary O, Chaligne R, Mazutis L. Nuclei extraction for single-cell RNAseq from frozen tissue using Singulator 100. protocols.io. 2022. [https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1](https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1) - Protocols.io. Nuclei Isolation for SnRNA-seq and SnATAC-seq from Frozen Fresh Human Retina Sample. [https://www.protocols.io/view/nuclei-isolation-for-snrna-seq-and-snatac-seq-from-bftmjnk6](https://www.protocols.io/view/nuclei-isolation-for-snrna-seq-and-snatac-seq-from-bftmjnk6) - Mazutis L, Masilionis I, Chaudhary O. Frozen tissue dissociation for single-nucleus RNA-Seq. protocols.io. [https://www.protocols.io/view/frozen-tissue-dissociation-for-single-nucleus-rna-5k5g4y6](https://www.protocols.io/view/frozen-tissue-dissociation-for-single-nucleus-rna-5k5g4y6) - Denisenko et al. (2020). Systematic assessment of tissue dissociation and storage biases in single-cell and single-nucleus RNA-seq workflows. *Genome Biology* 21, 130. - 10x Genomics (2023). Demonstrated Protocol: Dissociation of Mouse Tissues for Single Cell RNA Sequencing. CG000183. - Adam M, Potter AS, Potter SS. (2017). Psychrophilic proteases dramatically reduce single-cell RNA-seq artifacts: A molecular atlas of kidney development. Development 144(19): 3625-3632. DOI: 10.1242/dev.151142. - Guilliams et al. (2022). Spatial proteogenomics reveals distinct and evolutionarily conserved hepatic macrophage niches. *Cell* 185(2): 379-396. - Precision Cell Systems Application Note: Isolation of Viable Cells from Mouse Heart Tissue with the Singulator 100. P/N: 100-261-728. - Precision Cell Systems Application Note: Single-Cell Sequencing of Tumor-Infiltrating Lymphocytes in Lung Cancer: A Comparative Analysis Using the Singulator Platform. - Precision Cell Systems Application Note: Isolation of Mouse Skin Cells with the Singulator Platform. P/N: 100-261-837. - Precision Cell Systems Application Note: Sequencing Skin and Kidney Cells: Leveraging the Singulator 100 and Parse Biosciences' Evercode WT v2 Platform. P/N: 100-261-065. - Precision Cell Systems. Singulator Demonstrated Protocol: Cell Isolation from Intestine Tissue for Single Cell Sequencing. - Precision Cell Systems Application Note: Dissociating Tumor Tissue into Cells with Precision Cell Systems' Pan Tumor Reagent. P/N: 100-261-619. - Precision Cell Systems. Singulator Demonstrated Protocol: Nuclei Isolation and Cleanup from Frozen Tissue (Standard and Low Volume protocols). - Precision Cell Systems. 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NKX2-2 based nuclei sorting on frozen human archival pancreas enables the enrichment of islet endocrine populations for single-nucleus RNA sequencing. BMC Genomics. 2024;25:427. [https://link.springer.com/article/10.1186/s12864-024-10335-w](https://link.springer.com/article/10.1186/s12864-024-10335-w) - Whytock KL, et al. Protocol for flow cytometry-assisted single-nucleus RNA sequencing of human and mouse adipose tissue with sample multiplexing. STAR Protocols. 2024;5(1):102893. [https://pubmed.ncbi.nlm.nih.gov/38416649/](https://pubmed.ncbi.nlm.nih.gov/38416649/) - Nobs SP, et al. A Simple, Quick, and Partially Automated Protocol for the Isolation of Single Nuclei from Frozen Mammalian Tissues for Single Nucleus Sequencing. JOVE. 2023;(197):e65611. [https://www.jove.com/t/65611/a-simple-quick-partially-automated-protocol-for-isolation-single](https://www.jove.com/t/65611/a-simple-quick-partially-automated-protocol-for-isolation-single) - Slyper M, Porter CBM, Ashenberg O, et al. 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[https://pmc.ncbi.nlm.nih.gov/articles/PMC7971773/](https://pmc.ncbi.nlm.nih.gov/articles/PMC7971773/) - Drokhlyansky E, et al. The Human and Mouse Enteric Nervous System at Single-Cell Resolution. Cell. 2020;182(6):1606-1622. [https://www.sciencedirect.com/science/article/pii/S0092867420309946](https://www.sciencedirect.com/science/article/pii/S0092867420309946) - Lukowski SW, et al. Single-nuclei RNA-seq on human retinal tissue provides improved transcriptome profiling. Nature Communications. 2019;10:5743. [https://www.nature.com/articles/s41467-019-12917-9](https://www.nature.com/articles/s41467-019-12917-9) - Precision Cell Systems. Application Note: Sequencing Lung and Kidney Nuclei: Leveraging the Singulator 100 and Parse Biosciences Evercode Whole Transcriptome v2 Platform. P/N: 100-261-174. - Borcherding N, et al. Isolation and RNA sequencing of single nuclei from Drosophila tissues. STAR Protocols. 2022;3(3):101417. [https://www.sciencedirect.com/science/article/pii/S2666166722002970](https://www.sciencedirect.com/science/article/pii/S2666166722002970) - Precision Cell Systems. Application Note: Use of NIC+ with the Singulator 100 for Sub-20 mg Samples. NIC+ vs Standard cartridge yields across brain, kidney, lung, heart, breast tissues. - Precision Cell Systems. Application Note: Use of the Nuclei Debris Removal Stock Reagent. P/N: 100-261-392. - Precision Cell Systems. Application Note: Single-Nuclei Sequencing of Mouse Cerebellum Using the Singulator 100. - Precision Cell Systems. Application Note: FFPE Tissue Dissociation -- Singulator 200+ vs. Alternative Workflows: Evaluating Automated Nuclei Extraction for Mouse PDAC Tissues. - Romero R, Chu T, González-Robles TJ, et al. The neuroendocrine transition in prostate cancer is dynamic and dependent on ASCL1. Nature Cancer. 2024;5:1641-1659. DOI: 10.1038/s43018-024-00838-6. - Nadelmann ER, et al. 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[https://www.sciencedirect.com/science/article/pii/S2666166723007232](https://www.sciencedirect.com/science/article/pii/S2666166723007232) - Worthington Biochemical (2023). Tissue Dissociation Guide. [https://www.worthington-biochem.com/tissuedissociation/](https://www.worthington-biochem.com/tissuedissociation/) - Worthington Papain Dissociation System protocol. Catalog LK003150. - Miltenyi Biotec (2023). Adult Brain Dissociation Kit protocol. 130-107-677. [Back to all resources](/#library) ## Similar resources [Publication](https://elifesciences.org/articles/81090?__hstc=225964101.fe4380f775ce29a6a07134828a8c07b8.1742318073258.1743189924157.1743558253016.10&__hssc=225964101.27.1743558253016&__hsfp=1300058700&submissionGuid=d621ffa4-d176-4293-828a-2ca2f3c1f2e6) Singulator 100 Singulator 200 2025 ### Single-nuclei characterization of pervasive transcriptional signatures across organs in response to COVID-19 [View Publication](https://elifesciences.org/articles/81090?__hstc=225964101.fe4380f775ce29a6a07134828a8c07b8.1742318073258.1743189924157.1743558253016.10&__hssc=225964101.27.1743558253016&__hsfp=1300058700&submissionGuid=d621ffa4-d176-4293-828a-2ca2f3c1f2e6) [Protocol](https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1?__hstc=225964101.fe4380f775ce29a6a07134828a8c07b8.1742318073258.1743114390284.1743120525713.7&__hssc=225964101.13.1743120525713&__hsfp=1300058700&submissionGuid=cc1fb887-2fc0-423d-9124-6e7264b1d55f) Singulator 100 Singulator 200 2025 ### Nuclei extraction for single-cell RNAseq from frozen tissue using Singulator™ 100 [Read Protocol](https://www.protocols.io/view/nuclei-extraction-for-single-cell-rnaseq-from-froz-q26g74xzqgwz/v1?__hstc=225964101.fe4380f775ce29a6a07134828a8c07b8.1742318073258.1743114390284.1743120525713.7&__hssc=225964101.13.1743120525713&__hsfp=1300058700&submissionGuid=cc1fb887-2fc0-423d-9124-6e7264b1d55f) [App Note](https://precisioncellsystems.com/wp-content/uploads/2025/09/Research-Spotlight-Optimizing-Nuclei-Isolation-for-Reliable-snRNA-seq-A-Comparative-Study-Validates-the-SingulatorTM-Platform.pdf) Singulator 100 Singulator 200 2025 ### Optimizing Nuclei Isolation for Reliable snRNA-seq - 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