RNA sequencing of human primary myotubes from lean and obese subjects

Fasting muscle biopsy samples from the vastus lateralis were collected from lean (LN: BMI < 25 kg/m2) and obese (OB: BMI > 40 kg/m2) subjects. Primary myoblasts were isolated from the biopsy samples, maintained in vitro, and differentiated to myotubes. Overall design: Total RNA from three samples per group was isolated by Qiagen RNeasy kit and quality was assessed on a bioanalyzer (RIN>8 for each sample). Poly-A selected cDNA libraries were constructed and sequenced on an Illumina HiSeq2000 by Otogenetics Corp (Norcross, GA) producing at least 20 million 2X101 paired end reads per sample. Reads were aligned to the NCBI GRCh37 USCS hg 19 reference genome with TopHat. RefSeq annotations were used as the gene model and differential expression was assessed with Cufflinks, correcting for multiple comparisons. Gene enrichment and pathway analysis were conducted with the Database for Annotation, Visualization and Integrated Discovery (DAVID).