Single-Cell RNA-Seq of Purified Splenic T Cells

Thymus and spleens were processed as described previously (15 (link)). Briefly, they were dilacerated and treated with 1 mL RBC Lysis Buffer (ThermoFischer Scientific). Then cells were resuspended in PBS supplemented with 2% FBS. Splenic T cells were further purified using EasySep™ Mouse T Cell Isolation Kit (StemCell technologies). Each sample was labelled with a distinct TotalSeq™-A anti-mouse Hashtag reagent (BioLegend). The different samples were pooled, then loaded on a Chromium Chip B (10X Genomics), and cells were droplet-encapsulated with a Chromium Controller (10X Genomics). Single-cell cDNA synthesis and sequencing libraries were prepared with Chromium Single Cell 3’ v3 Library and Gel Bead kit (10X Genomics) according to manufacturer’s instructions. Libraries were sequenced using a Next-seq500 (Illumina) and the following parameters, Read1: 26 cycles, i7: 8 cycles, Read2: 57 cycles.