203 Fatty Acid Oxidation in PLN R14del Cardiomyopathy 8 systematic digital histological quantification of fibrosis and fatty tissue in trichrome stained slides was used to create schematic overviews showing mean fibrosis or adipose tissue in the PLN-R14del and control groups (Fig.S8B). If applicable, macroscopically visible regions of subepicardial fat or myocardial fibrofatty replacement were removed from frozen tissue blocks and 12-30 tissue slices of 10 µm thick were cut to achieve a comparable amount of myocardial tissue in the sequenced material. For all the other samples ten 10 µm thick frozen slices were collected. Chromatin H3K27ac immunoprecipitation and sequencing of human cardiac tissues. Chromatin was isolated from frozen cardiac tissues using the MAGnify™ Chromatin Immunoprecipitation System kit (Life Technologies) according to the manufacturer’s instructions. In brief, the obtained cardiac tissue was crosslinked with 1% formaldehyde and the crosslinking was stopped by adding 1.25 M glycine. Cells were lysed using the kit-provided lysis buffer and nuclei were sonicated using Covaris microTUBE (duty cycle 5%, intensity 2, 200 cycles per burst, 60s cycle time, 10 cycles). Sheared chromatin was diluted based on the expected number of isolated cells and was incubated with an anti-H3K27ac antibody (ab4729, Abcam) pre-coupled to magnetic beads for 2 hours at 4°C. Beads were extensively washed and crosslinking was reversed by the kit-provided reverse crosslinking buffer with 20 mg/mL Proteinase K. DNA was purified using ChIP DNA Clean & Concentrator kit (Zymo Research). Isolated DNA was additionally sheared, end repaired, sequencing adaptors were ligated and the library was amplified by PCR using primers with sample-specific barcodes according to our modification to the manufacturer’s recommendations.2 After PCR, the library was purified and checked for the proper size range and for the absence of adaptor dimers on a 2% agarose gel and sequenced on SOLiD Wildfire sequencer. Histone acetylation data processing. ChIP-seq peak identification: Sequencing reads were mapped against the reference genome (hg19 assembly, GRCh37) using the BWA package (-c, –l 25, -k 2, -n 10).3 Multiple reads mapping to the same location and strand have been collapsed to single reads and only uniquely placed reads were used for peak/region calling. Regions were called using Cisgenome 2.0 ( –e 150 -maxgap 200 –minlen 200).4 Next, to obtain a common reference, region coordinates from all PLN-R14del and control samples were stretched to at least 2000 base pairs and collapsed into a single common list. Overlapping regions were merged based on their outmost coordinates. Only the autosomal regions supported by at least 2 independent datasets were further analysed. Sequencing reads from each ChIP-seq library were overlapped with the common region list, to set the H3K27ac occupancy for every region-sample pair. Obtained regions were further analysed using 4 analyses. Detection of differentially acetylated regions: Regions with differential H3K27ac occupancy between PLN-R14del and control hearts were identified using DESeq2 standard settings
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