title: Transcriptomic analysis of PAX7+ cells isolated from injured Tibialis Anterior muscles of mice exposed to hypoxic versus normoxic atmosphere
design: Transcriptomic analysis of PAX7+ cells isolated from injured Tibialis Anterior muscles of mice exposed to hypoxic versus normoxic atmosphere
library_name: Norm4_s
library_construction_protocol: Seven days after cardiotoxin-induced muscle injury, Tibialis Anterior (TA) muscles from Tg:Pax7-nGFP mice exposed to normoxic (20%) or hypoxic (10%) environment were dissected, minced and incubated with a mix of 2.4U/ml Dispase II (Roche, #04942078001) and 100μg/ml collagenase A (Roche, #11088793001) in 4.5g/L glucose 1x DMEM (GibcoTM; #11995) at 37 °C on a shaking plate for 2 h. The muscle suspension was filtrated through 100-μm and 70-μm cell strainers and then spun at 50g for 10min at 4°C to remove large tissue fragments. Then, the supernatant was collected, filtered through a 40-μm cell strainer and washed by centrifugation at 700g for 10 min at 4°C. The final pellet has been resuspended in cold 4.5g/L glucose 1xDMEM supplemented with 0.2% bovine serum albumin (BSA, Sigma-Aldrich). The muscle cell suspension was used to sort 140 000 GFP+ MuSCs by fluorescence-activated cell sorting (FACS)-sorting using the GFP reporter (n=4 per atmospheric condition). Total RNA was extracted from GFP+ myogenic cells isolated from injured TA using RNAqueous™-Micro Kit RNA Isolation ™ (Invitrogen™, #AM1931). Library preparation and sequencing was performed by the Biomics platform at the Institut Pasteur. RNA preparation was used to build libraries using an Illumina Stranded mRNA library Preparation Kit (Illumina, USA) following the manufacturer's protocol. Quality control was performed on an Agilent BioAnalyzer