Transcriptomic analysis of C2C12 myotubes after adenoviral expression of Crebrf
Purpose: identify global changes in gene expression in C2C12 myotubes caused by an increase in Crebrf. Methods: Triplicate replicates of differentiated C2C12 myotubes expressing either Gfp or Crebrf for 3 days after transduction. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2. Results: Gene set enrichment analysis reveals oxidative phosphorylation and interferon response as the top up- and down-regulated gene sets, respectively, upon Crebrf expression.  The glycolysis gene set is also reduced.  These gene expression findings parallel our metabolic observations in Seahorse analysis. Conclusions: Our study indicates that CREBRF is a strong regulator of muscle metabolism in C2C12 myotubes Overall design: C2C12 myotubes were transduced by adenovirus encoding Gfp or Crebrf and RNA was isolated and analyzed by deep sequencing, in replicate, using Illumina HiSeq2000.