Chromium 10× single-cell 3′ mRNA sequencing of tumor-infiltrating lymphocytes

Chromium 10× 3′ V2 protocol is a 3′ end counting single-cell mRNA sequencing protocol that allows to process and sequence RNA from thousands of cells in parallel. Chromium10× by 10× Genomics is an emulsion-based device that enables to compartmentalize single cells along with sets of uniquely barcoded primers and reverse transcription reagents into nanoscale droplets that are used as reaction chambers to generate barcoded full-length cDNA from single cells. After RT reaction single-stranded barcoded cDNAs are pooled together and processed to generate sequencing libraries compatible with the standard Illumina platforms. Here we show in detail the main steps of the protocol applied to the analysis of tumor-infiltrating T lymphocytes (TILs). The main steps are cell preparation, cDNA synthesis, library construction, and sequencing. This protocol refers specifically to the CG00052_SingleCell3_ReagentKitv2UserGuide_RevD downloadable from 10× Genomics website (https://www.10xgenomics.com ) and does not substitute it. Always refer to this guide, paying attention to updates and revisions.