Profiling Active Chromatin by H3K27Ac ChIP-seq

Active status of chromatin was determined by histone 3 lysine 27 acetylation (H3K27Ac) levels using ChIP-seq. H3K27Ac ChIP assay was conducted with 5 μg of anti-H3K27Ac antibody (Active Motif, 39133) using the protocol described above. Sequencing libraries were prepared with 3 ng each of H3K27Ac ChIP DNA and input sample using SMARTer ThruPLEX DNA-seq kit (Takara, R400675). Libraries were sequenced with single-end (SE) 75 cycles on an Illumina Nextseq 500 system at the Broad Institute of Harvard and MIT and the reads were aligned to human reference genome hg19 using Burrows-Wheeler Alignment (BWA) tool^{44 (link)}. Genome-wide coverage was calculated after extending to 200 bases (approximate fragment size) and averaged over 25 bp windows using igvtools^{45 (link)}. Coverage was then normalized and scaled using RSeqC (http://rseqc.sourceforge.net/#normalize-bigwig-py). ChIP-seq peaks were called using MACS2 2.0.10.20120913.