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

Nat Commun. 2021 Jan 25;12(1):569. doi: 10.1038/s41467-020-20751-7.


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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Gene Expression Profiling / methods
  • High-Throughput Nucleotide Sequencing / methods*
  • Humans
  • Mice
  • RNA / genetics
  • RNA, Messenger / genetics
  • Reproducibility of Results
  • Sequence Analysis, RNA / methods*
  • Single-Cell Analysis / methods*
  • T-Lymphocytes, Cytotoxic / cytology
  • T-Lymphocytes, Cytotoxic / metabolism*


  • RNA, Messenger
  • RNA