Single-Cell RNA-Seq for Immune Profiling

Single-cell RNA-seq experiment was carried out as previously described.27 (link) Single-cell suspensions were obtained as described in the Tumor-infiltrating immune cell profiling section and were sorted using 4′,6-diamidino-2-phenylindole (DAPI) staining. Cells were then resuspended into single cells at a concentration of 1×10⁶ per mL in 1× PBS with 0.04% bovine serum albumin (BSA) for 10× genomics processing. Cell suspensions were loaded onto a 10× Genomics Chromium instrument to generate single-cell gel beads in emulsion. Approximately 5000–10,000 cells were loaded per channel. Single-cell RNA sequencing (scRNA-seq) libraries were prepared using the following Single Cell 3′ Reagent Kits: Chromium Single Cell 3′ Library & Gel Bead Kit v2 (PN-120237), Single Cell 3′ Chip Kit v2 (PN-120236) and i7 Multiplex Kit (PN-120262) (10× Genomics, Pleasanton, California, USA) following the Single Cell 3′ Reagent Kits V.2 User Guide (manual part #CG00052 Rev A). Libraries were run on an Illumina HiSeq 4000 system (SY-401–4001, Illumina) as 2×150 paired-end reads, one full lane per sample, for approximately >90% sequencing saturation.