GSM7430986: Pax7High_c; Homo sapiens; RNA-Seq
Human myoblasts and human MuRC were generated from six different biological replicates (N=6) and stained for Pax7 as previously described (Pax7 intra-cellular staining) in the presence of RNAse inhibitor (l/500, RNasin® Ribonuclease Inhibitors, Promega) in all buffers from the fixation step to the cell sorting step. Human myoblasts, MuRC-Pax7High and MuRC-Pax7Low subpopulations were then prepared for bulk RNA-sequencing according to the following steps: (1) flow cytometry cell sorting of human myoblasts Pax7+, of MuRC-Pax7High subpopulation and of MuRC-Pax7Low subpopulation (200,000 cells per condition) using a BD FACS Aria2 collected in FACS tube containing RNAse inhibitor and RNA later (2) RNA was extracted using either a high pure FFPE RNA Isolation Kit (Roche, cat#06650775001) or an RNeasy FFPE kit (Qiagen). Both kits were used following the manufacturer's protocol and kept at -80°C until use (3) RNA concentrations were obtained using Qubit RNA assay (Life Technologies, New York, USA). The quality of the RNA (RNA integrity number) was determined on the Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). (4) Sequencing was performed on the HiSeq 4000 unit (Illumina, San Diego, CA) using Illumina TruSeq RNA Exome (previously known as the Illumina TruSeq RNA Access Library Prep). Samples were sequenced as single-end 75 bp, and an average of 21,769,230 reads were generated for each sample. RNA-seq experiments were performed at the iGE3 Genomics Platform of the University of Geneva (http://www.ige3.unige.ch/genomics-platform. php). Illumina TruSeq RNA Exome