Well-Paired-Seq2: High-Throughput and High-Sensitivity Strategy for Characterizing Low RNA-Content Cell/Nucleus Transcriptomes

Abstract High-throughput single-cell RNA sequencing (scRNA-seq) is recognized as a powerful technology for disentangling the heterogeneity of cellular states. However, the Poisson-dependent cell capture and low sensitivity in scRNA-seq methods pose challenges for throughput and for samples with low RNA-content. Herein, to address these challenges, we developed Well-Paired-Seq2 (WPS2) based on size-exclusion and locally quasi-static hydrodynamic principles to realize high efficiency of cell utilization, single cell/bead pairing, and cell-free RNA removal. WPS2 exploits molecular crowding effect, tailing activity enhancement in reverse transcription, and homogeneous enzymatic reaction in the initial bead-based amplification to achieve 3116 genes and 8447 transcripts. With an average of ∼20,000 reads per cell, WPS2 detected 1420 more genes and 4864 more transcripts than our previous Well-Paired-Seq. Using WPS2, we overcame the Poisson limit for the capture of both cells and beads and accurately characterized transcriptomes of low RNA-content single cells and nuclei with high sensitivity. WPS2 was further applied to comprehensively profile transcriptomes from frozen clinical samples. We found that clear cell renal cell carcinoma (ccRCC) has a complex microenvironment, and that chromophobe renal cell carcinoma (chRCC) exhibits abundant copy number variations (CNVs). In addition, metanephric adenoma (MA) was characterized at single-cell level for the first time and some potentially specific markers were revealed. With the advantages of high sensitivity, high throughput, and high fidelity, we anticipate that WPS2 will be broadly applicable in basic and clinical research.