RNA-seq Library Preparation and Analysis

RNA-seq libraries were constructed as previously described^{84 (link)}. Briefly, first-strand cDNA was synthesized from oligo(dT) Dynabead-captured mRNA using SuperScript VILO cDNA Synthesis Kit (Invitrogen). Second-strand cDNA was synthesized using DNA polymerase I (New England Biolabs). cDNA was purified using Agencourt AMPure XP beads (Beckman Coulter). Nextera transposase (Illumina; 5 min at 55 °C) was used to simultaneously fragment and insert sequencing primers into the double-stranded cDNA. Transposition reactions were halted using QG buffer (Qiagen) and fragmented cDNA was purified on AMPure XP beads. Indexed sequencing libraries were generated by six cycles of PCR. Libraries were sequenced using 50-bp paired-end reads on one lane of an Illumina HiSeq 2000 instrument at the Duke Genome Sequencing and Analysis Core Resource. Reads were aligned to human RefSeq transcripts using Bowtie^{85 (link)}. The significance of differential expression of ^WTMyoD and ^VP64MyoD treated samples compared to untreated control samples, including correction for multiple hypothesis testing, was calculated using DESeq^{86 (link)}. Sequencing data has been deposited to the Gene Expression Omnibus, Accession code: GSE62448.