Antigen

Antigen Class

Histone

Antigen

H3K27ac

Cell type

Cell type Class

Cardiovascular

Cell type

Heart Ventricles

MeSH Description

The lower right and left chambers of the heart. The right ventricle pumps venous BLOOD into the LUNGS and the left ventricle pumps oxygenated blood into the systemic arterial circulation.

Sample

source_name

Mouse ventricle below left anterior descending artery ligation plane

strain

ICR/CD1

surgery performed at developmental age

Postnatal day 8

sample collected at post-surgical day

day 7

surgery type

MI

antibody

H3K27ac

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Each heart was dissected in ice-cold PBS and a transverse cut on ligation plane was made, separating it into above ligation plane (ALP) part and below ligation plane (BLP) part. BLP parts were flash-frozen with liquid nitrogen and stored in -80°C for RNA-Seq. The collected tissue samples were ground to powder in liquid nitrogen, and crosslinked with 2% formaldehyde in PBS for 30 min and quenched with 0.125M glycine for 10 min at room temperature. Crosslinked samples were then washed with cold PBS and further homogenized with a Dounce tissue grinder. Samples were collected by a brief spin, and treated with 10 mM Tris-HCl (pH 8.0), 10mM NaCl and 0.2% NP-40 for 30 min to collect nuclei. After nuclear extraction, chromatin was sheared on a Bioruptor Pico (Diagenode) for 20 cycles (30 sec on/ 30 sec off for each cycle) at 4°C in sonication buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 0.25% sarkosyl, 1 mM DTT, 1x cOmplete Protease Inhibitor Cocktail (Roche), and 1 mM PMSF, pH 8.0). After sonication, 1% of the sonicated chromatin from each sample was taken out as “input” samples. The remaining sonicated chromatin was evenly split for H3K27ac or H3K27me3 ChIP. Sonication buffer was added to make the final volume to 1 mL. NaCl was then added to a final concentration of 300 mM for each sample. 1 µg H3K27ac antibody (Diagenode, C15410196) or H3K27me3 (Diagenode, C15410195) was added to each sample and incubated at 4°C overnight with gentle rotation. The next day, 30 µL of pre-washed Dynabeads Protein G (Invitrogen, 10004D) was added to each sample for a two-hour incubation. After that, the beads were washed twice with 1 mL RIPA 0 buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, pH 8.0), twice with 1 mL RIPA 0.3 buffer (0.1% SDS, 1% Triton X-100, 10 mM Tris-HCl, 1 mM EDTA, 0.1% sodium deoxycholate, 300 mM NaCl, pH 8.0), twice with 1 mL LiCl wash buffer (250 mM LiCl, 0.5% IGEPAL CA-630, 0.5% sodium deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.0), and finally twice with 1 mL TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). For each ChIP sample or input sample, 100 uL of SDS elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.0) was added and incubation was done at 65°C overnight on a ThermoMixer (Eppendorf) at 1000 rpm. The next day, supernatant was collected and further treated with 0.5 µg RNaseA (Sigma, 11119915001) for 30 min at 37°C, followed by 20 µg Proteinase K (NEB, P8107S) treatment at 37°C for 2h. DNA was recovered using MinElute PCR Purification Kit (QIAGEN, 28004) according to manufacturer's protocol. ChIP-Seq libraries were generated using NEBNext Ultra II DNA Library Prep Kit for Illumina (NEB, E7645L) according to manufacturer's protocol.

Sequencing Platform

instrument_model

NextSeq 500

mm10

Number of total reads

26897877

Reads aligned (%)

98.1

Duplicates removed (%)

13.1

Number of peaks

45797 (qval < 1E-05)

mm9

Number of total reads

26897877

Reads aligned (%)

98.1

Duplicates removed (%)

13.2

Number of peaks

45811 (qval < 1E-05)