H3K27ac chromatin immunoprecipitation and sequencing (ChIP-seq)

We performed chromatin immunoprecipitation and sequencing (ChIP-seq) using the H3K27ac mark to study the differences of the histone acetylome between patient and control samples as previously described [10]. To study the differences of the histone acetylome between patient and control samples, we performed chromatin immunoprecipitation and sequencing (ChIP-seq) using the H3K27ac mark. Briefly, all cardiac samples were sectioned at a thickness of 10 µm, and chromatin was isolated using the MAGnify™ Chromatin Immunoprecipitation System kit (Life Technologies) according to the manufacturer’s instructions. The anti-histone H3K27ac antibody (ab4729, Abcam) was used for immunoprecipitation. Captured DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). Libraries were prepared using the NEXTflex™ Rapid DNA Sequencing Kit (Bioo Scientific). Samples were PCR amplified, checked for the proper size range and absence of adaptor dimers on a 2% agarose gel, and barcoded libraries were sequenced 75-bp single end on an Illumina NextSeq500 sequencer. Sequencing reads were mapped against the reference genome (hg19 assembly, NCBI37) using the BWA package (mem –t 7 –c 100 –M –R)1. Multiple reads mapping to the same location and strand were collapsed to a single read, and only uniquely placed reads were used for peak calling. Peaks/regions were called using Cisgenome 2.02 (–e 150 -maxgap 200 –minlen 200). Region coordinates from all samples were stretched to at least 2000 base pairs and collapsed into a single common list. Overlapping regions were merged based on their coordinates. Only regions supported by at least 2 independent datasets were further analyzed. Autosomal sequencing reads from each ChIP-seq library were overlapped back with the common region list to set the H3K27ac occupancy for every region-sample pair. Differentially acetylated regions between HCM and control hearts were identified using DESeq2 under the default setting in the Galaxy environment [44].