ChIP-exo Profiling of MEF2A Transcription Factor

Note that 15 × 10⁶ C2C12 (48 h DM) and 8 × 10⁶ primary rat CMs were prepared for ChIP-exo as follows: Cells were washed with 1× PBS and treated with 37% formaldehyde (Sigma-Aldrich) for 15 min at 37°C. The cell pellet was isolated similar to ChIP-qPCR as previously described (35 (link)). DNA was sonicated to ∼250 bp in length. Cross-linked chromatin was sent to Peconic Genomics with 5 μg anti-MEF2A (Santa Cruz) and Rabbit IgG (Millipore). Peconic Genomics completed ChIP-exo as previously described (31 (link)) and the resulting samples were sequenced on the Illumina HiSeq 2000 platform. Illumina CASAVA software was used for base calling and sequencing reads were aligned to the mm10 (MBs) or rn5 (CMs) genome assembly using BWA 0.5.9 (36 (link)). Raw data were filtered for a quality score of 37 using SAMtools (37 (link)), and duplicates were removed using Picard (http://picard.sourceforge.net/). MACS 1.4.2 was used to do peak calling analysis (38 (link)). To identify MEF2A target genes in skeletal and cardiac muscle corresponding to peak location, MEF2A enrichment peaks identified in MACS were converted to mm9 using UCSC LiftOver (39 (link)).