HyPR-seq: Single-cell quantification of chosen RNAs via hybridization and sequencing of DNA probes

Abstract

Single-cell quantification of RNAs is important for understanding cellular heterogeneity and gene regulation, yet current approaches suffer from low sensitivity for individual transcripts, limiting their utility for many applications. Here we present Hybridization of Probes to RNA for sequencing (HyPR-seq), a method to sensitively quantify the expression of up to 100 chosen genes in single cells. HyPR-seq involves hybridizing DNA probes to RNA, distributing cells into nanoliter droplets, amplifying the probes with PCR, and sequencing the amplicons to quantify the expression of chosen genes. HyPR-seq achieves high sensitivity for individual transcripts, detects nonpolyadenylated and low-abundance transcripts, and can profile more than 100,000 single cells. We demonstrate how HyPR-seq can profile the effects of CRISPR perturbations in pooled screens, detect time-resolved changes in gene expression via measurements of gene introns, and detect rare transcripts and quantify cell type frequencies in tissue using low-abundance marker genes. By directing sequencing power to genes of interest and sensitively quantifying individual transcripts, HyPR-seq reduces costs by up to 100-fold compared to whole-transcriptome scRNA-seq, making HyPR-seq a powerful method for targeted RNA profiling in single cells.

Current Methodological Challenges of Single-Cell and Single-Nucleus RNA-Sequencing in Glomerular Diseases

“…Some studies exclusively performed scRNA-seq on magnetically isolated glomeruli from mouse kidneys and all were successful in isolating podocytes. 11,32,[40][41][42][43] In unsorted renal tissue from mice, all seven snRNA-seq studies 9,33,34,[36][37][38][39] versus half (eight of 16) of the scRNA-seq studies 33,34,45,52,[55][56][57][58] were able to isolate podocytes (Supplemental Table 5). Almost all successful scRNA-seq studies analyzed .20,000 cells in total.…”

Light-Seq: light-directed in situ barcoding of biomolecules in fixed cells and tissues for spatially indexed sequencing

“…We expect that Light-Seq's sensitivity can be further improved with optimization of the in situ RT and barcoding, such as by protease treatment, antigen retrieval or changes to fixation/permeabilization conditions 21,28,39,60 , use of targeted ISH probes 61 and targeted ribosomal RNA depletion. These improvements, combined with the flexibility of custom photomasks, could ultimately enable profiling of single cells or subcellular compartments with higher efficiency.…”

SnFFPE-Seq: towards scalable single nucleus RNA-Seq of formalin-fixed paraffin-embedded (FFPE) tissue

“…For FFPE samples with heavily degraded, short RNA fragments, random primers 20 or polyadenylation of short RNA sequences with SMART-Seq-total 28 may improve the capture rate. Furthermore, our nucleus extraction method can be coupled to multiple other profiling methods, including multiplexed antibody-based detection of proteins 12 or targeted mutation profiling 29,30 . Further optimization of tissue-specific snFFPE-Seq protocols combined with emerging spatial transcriptomics techniques for FFPE 8,31,32 and new computational methods that tackle sparsity should significantly enhance our understanding of the functional organization and interactions of cells in tissues, especially in disease.…”