Changes in WT C2C12 Mouse Muscle Myoblast gene expression throughout differentiation.

Genesis of skeletal muscle relies on the differentiation and fusion of mono-nucleated muscle progenitor cells into the multi-nucleated muscle fiber syncytium. The temporally-controlled cellular and morphogenetic changes underlying this process are initiated by a series of highly coordinated transcription programs. Ultimately, highly specific coordination of these transcription programs is critical for their masterfully timed transitions, which in turn facilitate the intricate generation of skeletal muscle fibers from a naïve pool of progenitor cells. This work aims to categorize changes of gene expression in vitro in C2C12 mouse muscle myoblasts while they begin to form these multinucleated myotubes. Overall design: Total RNA was isolated from n=3 cultured C2C12 myoblasts at D0 and D5 using Tri-Reagent according to the manufacturer's instructions (Sigma Aldrich). RNA quality was accessed using a 2100 Bioanalyzer System (Agilent), and samples with RNA Integrity Number (RIN) greater than 8.0 were sequenced. Illumina RNA-Seq was performed by the Genomics Services Laboratory (GSL) at Nationwide Children's Hospital (Columbus, OH). Quality assessment of the RNA-Seq data was performed using NGS-QC-Toolkit (v 2.3.3) with default settings (Patel & Jain 2012). Quality filtered reads were aligned to the mouse reference genome GRCm38 (mm10) using the Tophat2 (v 2.0.0) aligner with default settings (Kim et al. 2013). The R package DESeq2 (v 1.6.3) was used for differential gene expression analysis (Love et al. 2014). Read counts obtained from featureCounts were normalized by taking the median of each gene count across samples and dividing each sample gene count by the relative ratio of library sizes between the calculated median and sample size (Liao et al. 2014). The average normalized expression values of the samples were used to calculate fold change and P values.