Tailoring the resolution of single-cell RNA sequencing for primary cytotoxic T cells

Single-cell RNA sequencing in principle offers unique opportunities to improve the efficacy of contemporary T-cell based immunotherapy against cancer. The use of high-quality single-cell data will aid our incomplete understanding of molecular programs determining the differentiation and functional heterogeneity of cytotoxic T lymphocytes (CTLs), allowing for optimal therapeutic design. So far, a major obstacle to high depth single-cell analysis of CTLs is the minute amount of RNA available, leading to low capturing efficacy. Here, to overcome this, we tailor a droplet-based approach for high-throughput analysis (tDrop-seq) and a plate-based method for high-performance in-depth CTL analysis (tSCRB-seq). The latter gives, on average, a 15-fold higher number of captured transcripts per gene compared to droplet-based technologies. The improved dynamic range of gene detection gives tSCRB-seq an edge in resolution sensitive downstream applications such as graded high confidence gene expression measurements and cluster characterization. We demonstrate the power of tSCRB-seq by revealing the subpopulation-specific expression of co-inhibitory and co-stimulatory receptor targets of key importance for immunotherapy.