ChIP-seq Analysis of BRD4 and H3K27ac

CHL-1 cells were treated for 4 h with 500 nM BAY 1238097 or OTX-015 prior to cross-linking and harvest. One 15 cm cell culture dish with 80% confluent cells was used per immunoprecipitation. ChIP DNA preparation for sequencing was carried out as described previously^{38 (link)} with the following changes: cross-linked chromatin was sheared to 200–800 base pair fragments using sonication (30 s on/30 s off, high power, 2 × 7.5 min with a water bath change between the two cycles) in a Bioruptor sonicator UCD-200 (Diagenode, Liège, Belgium). About 1.5 μg antibodies (anti-BRD4 (A301-985A100, Bethyl Labs, Montgomery, TX, USA); anti-H3K27ac (Diagenode, C15410174)) were used per immunoprecipitation. DNA was purified using a PCR purification kit (Qiagen), followed by library preparation and genome-wide sequencing. In brief, samples were analyzed on a 2100 bioanalyzer using the DNA high sensitivity assay. Concentration was determined using a High Sensitivity DNA Kit. End repair and phosphorylation, and ligation of TrueSeq index adapter and amplification before subjection to sequencing (HiSeq Sequencing System) were done according to the TrueSeq ChIP Sample Preparation Guide (Illumina, Munich, Germany). Sequencing data are available in the GEO database with the accession number GSE95585.