title: ChIP seq of human left ventricle (input)
design: Chromatin immuno-precipitations were performed as previously described  with some modifications. Briefly, frozen left ventricle tissue was pulverized with a mortar and pestle, resuspended in PBS, and cross-linked with 1% formaldehyde at room temperature for 10 min. Chromatin was sonicated to obtain fragments with an average size ranging between 100-600 bp. Chromatin was incubated for 2h at 4 oC with 5 g of Active Motif H3K27ac antibody (cat# 39133, lot 01613007). Protein A and G Dynabeads (Invitrogen) were then added to this chromatin/antibody mixture for 30 minutes at 4 oC. Immuno-complexes were sequentially washed. The protein/DNA complexes were eluted in an SDS buffer (1% SDS, 50 mM Tris pH 8.0, 10 mM EDTA) at 37 oC for one hour. Samples were treated with Proteinase K at 37 oC and reverse-crosslinked overnight. Finally, the DNA was purified on Zymo ChIP clean and concentrate columns (Zymo Research), and the quality was assessed on the Agilent bioanalyzer. The ChIP-seq libraries were prepared using the Illumina TruSeq library preparation kit followed by pooling of libraries. Libraries were pooled and sequenced via single end 50bp reads on a HiSeq 2500 (6 libraries per lane) or HiSeq 4000 (8 libraries per lane)(Illumina).