GSM4680545: Pre-graft_3_EOM_500cells_Exp2_RNAseq; Mus musculus; RNA-Seq
Tibialis anterior and extraocular muscles were dissected and placed into cold DMEM (ThermoFisher, 31966). Muscles were then manually chopped with scissors and put into a 15 ml Falcon tube containing 10 ml of DMEM, 0.08% collagenase D (Sigma, 11 088 882 001), 0.1% trypsin (ThermoFisher, 15090), 10 µg/ml DNase I (Sigma, 11284932) at 37°C under gentle agitation for 25 min. Digests were allowed to stand for 5 min at room temperature and the supernatants were added to 5 ml of foetal bovine serum (FBS; Gibco) on ice. The digestion was repeated 3 times until complete digestion of the muscle. The supernatants were filtered through a 70-µm cell strainer (Miltenyi, 130-098-462). Cells were spun for 15 min at 515g at 4°C and the pellets were resuspended in 1 ml cold freezing medium (10% DMSO (Sigma, D2438) in FBS) for long term storage in liquid nitrogen or processed directly through FACS-isolation for transplantations. Before isolation by FACS, samples were thawed in 50 ml of cold DMEM, spun for 15 min at 515g at 4°C. Pellets were resuspended in 300 µl of DMEM 2% FBS 1 µg/mL propidium iodide (Calbiochem, 537060) and filtered through a 40-µm cell strainer (BD Falcon, 352235). Viable muscle stem cells were isolated based on size, granulosity and GFP intensity using a MoFlo Astrios cell sorter (Beckmann Coulter). Cells were collected in 5 µl cold RLT Plus buffer (Qiagen, 1053393) containing 1U/µl RNAse inhibitor (Ambion, AM2694), flash-frozen on dry ice and stored at -80°C, or in cold DMEM 2% FBS. RNA was separated from cell lysates and processed into libraries using the G&T method as described in Macaulay et al., 2016. DNA was also processed into libraries using the bulk protocol described in Clark et al., 2017. Bisulfite-seq libraries were sequenced on an Illumina HiSeq-2500 with 125bp paired end reads and RNA-seq libraries were sequenced on an Illumina HiSeq-2500 with 75bp paired end reads.
GEO Accession
GSM4680545