Ronan Chaugnet
Director of the Single-Cell Analytics Innovation Lab (SAIL) at Memorial Sloan Kettering Cancer Center. Ronan runs a hybrid research lab and core facility that develops single-cell methods and processes samples for researchers across MSK and other institutions. His team works with fresh, frozen, and FFPE tissue, and their monthly sample volume has grown substantially over the past several years.
Summary
Ronan Chaugnet from Memorial Sloan Kettering shares several years of head-to-head benchmarking data comparing nuclei extraction platforms for FFPE tissue. The Singulator 200+ and Miltenyi GentleMACS both outperformed manual approaches across glioblastoma, liver, lung, pancreas, and tumor samples, with the Singulator 200+ standing out for its fully automated, enzyme-free workflow. Ronan also walks through three methods his lab has built on top of automated FFPE extraction: 10X Genomics FLEX for high-quality gene expression, PERFF-Seq for sorting rare cell populations by RNA markers, and GIFT-Seq for detecting 600+ mutations at single-cell resolution from archival tissue.
What You'll Learn
- How the major nuclei extraction platforms actually compare, with UMI recovery and cell type data from the Singulator 200+, Miltenyi GentleMACS, and 10X Genomics across glioblastoma, liver, lung, pancreas, and PDAC tumor tissue
- What makes the Singulator 200+ the preferred choice at MSK, including on-instrument deparaffinization, enzyme-free processing, and why Ronan's entire wet lab team rated it the most hands-off platform they tested
- Two new methods that depend on reliable FFPE nuclei extraction: PERFF-Seq for sorting rare cells by RNA markers (no antibodies needed), and GIFT-Seq for genotyping 600+ mutations alongside whole-transcriptome profiling
Key Takeaways
FFPE tissue is now fully accessible for single-cell genomics
The 10X Genomics FLEX kit uses short probes instead of poly-dT capture, so it works even when RNA is fragmented by formalin fixation. Ronan's lab now routinely gets over 6,000 UMIs per nucleus from clinical FFPE samples that have been in storage for years, with all expected cell types showing up in clean clusters. That means the massive FFPE archives sitting in pathology departments are no longer off-limits for single-cell work.
Automated extraction delivers the highest and most reproducible nuclei quality
Across glioblastoma, liver, lung, pancreas, and tumor tissue, the Singulator 200+ and Miltenyi GentleMACS both hit close to the target 10,000 nuclei post-sequencing and produced the highest UMI counts. The 10X Genomics protocol, which incubates with Liberase enzyme at 60 degrees, recovered only a few thousand nuclei from the same target. The difference comes down to how much you stress the already-fragmented FFPE RNA during extraction. The Singulator skips enzymatic digestion entirely, which keeps more of the remaining RNA intact.
The Singulator 200+ is the most hands-off platform for FFPE nuclei extraction
The Singulator 200+ runs deparaffinization on-instrument with a xylene-free reagent, so you don't need a chemical hood. There are no manual buffer additions. You load your tissue into the cartridge, press play, and walk away. Every person on Ronan's wet lab team rated it the easiest platform they tested. As Ronan put it: "If you do it today, tomorrow, and in three days, you will get the same results." For a core facility where different people are running the instrument each day, that consistency matters.
PERFF-Seq enables rare cell enrichment from FFPE using RNA markers
PERFF-Seq, developed by Caleb Lareau and Tsion Abay with MSK's SAIL lab, solves a problem that has frustrated researchers for years: how do you sort nuclei for a specific cell population when there are no surface proteins left to target? The answer is RNA FISH probes. You tag nuclei based on which genes they express, then FACS sort them the same way you would with antibodies. It works on fresh, frozen, and FFPE tissue, supports AND/OR/NOT gating logic, and can even target long non-coding RNAs like Xist. The full protocols are on Protocols.io.
GIFT-Seq detects mutations at single-cell resolution from archival FFPE
GIFT-Seq (Genotyping in Fixed Transcriptome) lets you detect SNVs and get whole-transcriptome gene expression from the same FFPE nuclei in the same experiment. Custom probes are spiked into the FLEX assay, leaving small gaps at mutation sites that get filled and sequenced. Ronan's team has validated this on 600+ loci with over 90% accuracy and under 2% contamination. On clinical glioblastoma FFPE, they genotyped EGFR mutations in roughly half the nuclei. The method was developed in collaboration with 10X Genomics.
Sample preparation decisions have outsized impact on data quality
The cell types you recover depend more on how you extract nuclei than on which sequencing assay you use. Enzymatic digestion at high temperatures measurably degrades RNA in FFPE. Shorter fixation times preserve more signal. DV200 is a useful quality metric, but Ronan has seen samples with DV200 around 30-40% still produce usable data. And with FLEX Apex bringing the per-sample cost down, his advice for borderline clinical samples is straightforward: just try it.
