GSM8191409: 322761_1-WT; Mus musculus; RNA-Seq
For isolation of cells for scRNAseq, skeletal muscle was collected and minced in a petri dish on ice using scissors and a surgical blade. Minced samples were transferred into a digestion buffer (4 mL digestion buffer per 100 mg of muscle) containing 2 mg/ml Dispase II (D4693 Sigma-Aldrich) and 2 mg/ml Collagenase IV (17104019 Thermo Fisher Scientific) in HBSS buffer (14025100 Thermo Fisher Scientific) at 37°C for 25 minutes, with gentle shaking every 5 minutes. After digestion, the reaction was stopped by adding an equal volume of 20% heat-inactivated FBS (10500064 Thermo Fisher Scientific) in HBSS (14025100 Thermo Fisher Scientific) and the suspension was passed through a series of 70-µm cell strainers (#352360, Corning, New York, USA) and 40-μm cell strainers (#352350, Corning, New York, USA) to remove tissue debris. Cell suspension was centrifuged at 450 g for 5 minutes at 4°C and the supernatant was discarded. The pellet was resuspended 2% FBS (10500064 Thermo Fisher Scientific) in DPBS (14190250 Thermo Fisher Scientific) and incubated with anti-CD31 and anti-CD45 antibodies for 15 minutes on ice. Thereafter, cells were washed and subjected for sorting. Metabolically active (Calcein+) human muscle mononuclear cells were FACS sorted and loaded into the 10X Chromium Next GEM Chip G, aiming the recovery of ~10000 cells per sample. scRNAseq libraries were generated according to 10X Chromium Next Gem Single Cell 3' Reagent Kits v3.1 (CG000204 Rev D) and sequenced on the Illumina Novaseq 6000 system