Single-cell RNA-seq Analysis Pipeline

Single-cell suspensions were counted using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter, Brea, CA, USA) and ~5000 cells were loaded into the 10X Genomics Chromium system to generate Gel Beads-In-Emulsion (GEMs). Reverse transcription was performed using Chromium Single Cell 3’ Reagent Kits (v2, 10X Genomics, Pleasanton, CA, USA). Standard Illumina sequencing primers and a unique i7 Sample index were added to each cDNA for library construction. Libraries were sequenced using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 × 2 paired-end reads on an Illumina HiSeq 4000 using HiSeq 3000/4000 sequencing kit and HCS v3.3.52 collection software. Base-calling was performed using Illumina’s RTA version 2.7.3. Cell Ranger (v3.0) (10X Genomics) was used for alignment to the mouse genome version mm10. R (v3.6.0) and Seurat (v3.1.3)^{35 (link)} were used for further analysis following the standard workflow. For quality control and filtering, cells with detected genes fewer than 200 or >8000, or with mitochondrial gene content >50% were excluded. For Ximerakis’s scRNA-seq dataset, R (v4.0.3) and Seurat (v4.0.2) were used. Gene set enrichment analysis was performed using software GSEA (v4.0.3)^{53 (link)}. The interactive website for scRNA-seq data was generated using ShinyCell (v2.1.0)⁵⁴ .