Single-cell RNA-seq protocol for immune cell analysis

scRNA-seq was performed as described^{79 (link)}. Briefly, single-cell suspensions from sorted live CD45+ cells were loaded on a GemCode Single Cell instrument (10x Genomics) to generate single-cell beads in emulsion and scRNA-seq libraries were prepared using the Chromium Single Cell 3′ Reagent Kits (v2), including Single Cell 3′ Library & Gel Bead Kit v2 (120237), Single Cell 3′ Chip Kit v2 (PN-120236), and i7 Multiplex Kit (120262) (10x Genomics) following the Single Cell 3′ Reagent Kits (v2) User Guide. Single-cell barcoded cDNA libraries were quantified by quantitative PCR (Kappa Biosystems) and sequenced on an Illumina NextSeq 500 (Illumina). Read lengths were 26 bp for read 1, 8 bp for i7 index, and 98 bp for read 2. Cells were sequenced to greater than 50,000 reads per cell as recommended by manufacturer.