Single-cell RNA-seq Library Preparation

Single-cell suspensions were achieved as described above and sorted using DAPI staining. Cells were then resuspended into single cells at a concentration of 1 × 10⁶ per mL in 1X PBS with 0.4% BSA for 10x genomics processing. Cell suspensions were loaded onto a 10x Genomics Chromium instrument to generate single-cell gel beads in emulsion (GEMs). Approximately 5,000 to 10,000 cells were loaded per channel. 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) (10x Genomics, Pleasanton, CA, USA) as previously described (67 (link)), and following the Single Cell 3′ Reagent Kits v2 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.