Single-cell skeletal muscle satellite cells and endothelial cells during homeostasis and regeneration

We performed single cell RNA sequencing on muscle satellite cells (MuSCs) and endothelial cells (ECs) isolated from mouse hind limb muscle from both basal condition and 3-days post CTX injury to look at both quiescent and activated SCs from mice expressing genetic reporters (Pax7CreERT2:R26RtdT:Flk1GFP) by FACS. We could reliably find injury and activated SC as judged by side and forward scatter. We FACS isolated cells from both days separately and spiked in 20% of the ECs into the SCs, and performed single cell RNA-seq for each time point. We performed extensive sequencing averaging more than 300K read/cell. We were able to align the data and overlap the basal and injured cells and able to deconvolve the quiescent SCs from the activated and more differentiated MuSCs, ECs and contaminating cell types from gene signatures. Overall design: Cells for single cell sequencing were obtained from Pax7tdT:Flk1GFP mice hind limb muscles following enzymatic digestion as previously described (Liu et al., 2015). Dead cells were excluded from the analysis using ZombieNIR (Biolegends, 423105). TdTomato+ and GFP+ cells were sorted individually and then 20% of GFP+ cells were spiked into 80% TdTomato+. We loaded ~5,000 cells into 1 channel of the Chromium system for each of these samples and prepared libraries according to the manufacturer's protocol using version 2.0 chemistry (10x Genomics). Following capture and lysis, we synthesized cDNA and amplified for 12 cycles as per the manufacturer's protocol (10X Genomics). The amplified cDNA was used to construct Illumina sequencing libraries that were each sequenced on one lane of an Illumina HiSeq 2500 machine. We used Cell Ranger 2.0 (10X Genomics) to process raw sequencing data. This pipeline converted Illumina basecall files to fastq format, aligned sequencing reads to the mm10 transcriptome using the STAR aligner, quantified the expression of transcripts in each cell using Chromium barcodes.