PERFF-seq – transcript-specific enrichment enables profiling rare cell states via scRNA-seq

In biology, each cell tells its own unique story—a narrative shaped by its gene expression profile and molecular interactions. Yet, uncovering these individual narratives within a sea of cellular diversity has long been a daunting task. Enter single-cell genomics—a revolutionary technology that promises to unravel the complex web of cellular heterogeneity with unprecedented precision. However, traditional methods of cell sorting have posed significant challenges, particularly when it comes to isolating rare cell populations with specific marker transcripts.

In a groundbreaking study, a team led by researchers at Memorial Sloan Kettering Cancer Center have developed a novel approach to overcome these limitations—Programmable Enrichment via RNA Flow-FISH by sequencing (PERFF-seq). This cutting-edge assay enables the precise profiling of subpopulations from complex cellular mixtures based on the presence or absence of specific RNA transcripts.

Rationale and development of PERFF-seq

(a) Schematic of the PERFF-seq assay. Target RNA(s) are bound by pairs of adjacent initiator probes that ensure specificity. Hairpin amplifiers unzip and hybridize iteratively to generate strong fluorescent signal and enable FACS prior to single-cell profiling with the droplet-based scRNA-seq Flex kit. (b) Knee plot of cells profiled with standard Flex versus HCR-FlowFISH sorted cells. (c) Fraction of reads fully mapping (blue) or half-mapping (grey) to the reference probe set. (d) Bioanalyzer traces highlighting expected product size of the full probe (∼260 bp; blue) and half probe (∼190 bp; grey) for a high-quality Flex library. (e) Same as (d) but for the FlowFISH → Flex v0 experiment. (f) Experiments identifying the HCR polymer as the corrupting agent for data quality. (g) Conditions screened for polymer stripping, including DNase and formamide treatments. (h) Sorting buffer components analyzed to improve data quality. (i) UMIs (top) and genes (bottom) detected per cell comparing initial FlowFISH → Flex v0 experiment, from (b) to the final PERFF-seq library, L, from panel (h). Values plotted in c and f–i represent overall library values for a single replicate.

The beauty of PERFF-seq lies in its versatility and scalability. By leveraging RNA-based cytometry followed by high-throughput scRNA-seq, researchers can now systematically enrich for rare cell populations identified by one or more marker transcripts. This groundbreaking method opens new avenues for studying cellular diversity across a range of biological contexts.

In their study, the researchers demonstrated the efficacy of PERFF-seq across diverse immune populations and even in challenging tissue samples such as fresh-frozen and formalin-fixed paraffin-embedded brain tissue. By providing a rational and programmable approach to cell sorting, PERFF-seq empowers researchers to unlock the full potential of single-cell genomics.

Abay T, Stickels RR, Takizawa MT, Nalbant BN, Hsieh YH, Hwang S, Snopkowski C, Yu KKH, Abou-Mrad Z, Tabar V, Ludwig LS, Chaligné R, Satpathy AT, Lareau CA. (2024) Transcript-specific enrichment enables profiling rare cell states via scRNA-seq. bioRXiv [online preprint]. [article]

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