RNA-seq analysis of CD8+ T cells

RNA from 0.25 to 1x10⁶ CD8⁺ T cells was isolated with Nucleospin RNA extraction kits (Macherey Nagel, catalog #740955.250); libraries were prepared using an Illumina TruSeq Library Kit and sequenced with an Illumina NovaSeq instrument. Reads were aligned on reference genomes (mm10 for mouse data, GRCh38 for human data) using the STAR universal RNA-seq aligner; DEGs were calculated with DESeq2. Heatmaps were created with Morpheus (Morpheus (broadinstitute.org)). For functional enrichment analyses, we used the enricher function (default parameters) from the clusterProfiler package v4.2.2 to perform hypergeometric tests for functional enrichment analysis. Only down-regulated genes were used as the input, and the universe/background was defined as all detected genes in our RNA-Seq. The human hallmark, C2 and GOBP (C5:BP) gene sets were retrieved from the Molecular Signatures Database [MSigDB (20 (link))] using the msigdbr function and package v7.4.1 Finally, we applied the Benjamini-Hochberg method to control false discoveries in multiple hypothesis testing.