GSM8942232: Activated mouse satellite cells (ASCs) from injured tibialis anterior muscles. Pax7+ (GFP+) sorted cells.; Mus musculus; RNA-Seq
Myoblasts and MPCs were collected by trypsinization and filtered through a 40um cell strainer. Cells were resuspended in PBS at a concentration of 1000cells/ul before proceeding with the Chromium Next GEM Single cell 3' v3.1 protocol. fiSCs and ASCs were retrieved from limb muscles or tibialis anterior muscles, respectively. In brief, pooled muscles were finely minced, resuspended and washed in PBS. Then the slurry was digested in Collagenase II for 1.5h , washed and filtered through 100um cell strainer. Digested muscles were briefly placed in LCK buffer, filtered through a 40um cell strainer and prepared for FACS-sorting. GFP+ sorted cells were centrifuged and counted before proceeding the the Chromium Next GEM Single cell 3' v3.1 protocol. All libraries were prepared according to the Chromium Next GEM Single cell 3' v3.1 protocol and sequenced on the Novaseq 6000 with paired ends. In brief, cells were mixed with barcoded single cell 3' gel beads and a master mix to generate GEMs. Then, barcoded cDNA is generated from polyadenlyated mRNA within GEMs. After, barcoded cDNA is amplified via PCR. Finally, cDNA is fragmented, end-repaired, ligated with adapters and amplified with sample index PCR.