ChIP Assay for Acetylated Histone H3 in Heart Tissue

As described in our previous study [15 (link)], EpiQuik™ Tissue ChIP Kit (P-2012; Epigentek Group Inc., Farmingdale, NY, USA) was used to perform the ChIP assay according to the manufacturer’s protocol. In brief, the c-Jun antibody (1 μg) or 1 μl of normal mouse IgG (as a negative control) was used to pre-coat the assay wells. Meanwhile, 30 mg of heart tissue was cut into little pieces and cross-linked with 1% formaldehyde. The cross-link was stopped by glycine solution (1.25 M). After tissue disaggregation and the nuclei isolation, the DNA was sheared by sonication (S-450-Dwithmicro-tip probe; Emerson Industrial, St. Louis, MO, USA) with 5 pulses of 20 s each separated by a 40 s rest on ice (output control: 2). After centrifugation, 5 μl of the diluted supernatants were used as input DNA. The other diluted supernatant (100 μl) was added to the acetylated histone H3 antibody-coated wells followed by incubation at room temperature for 60 min. ChIP-enriched DNA fragments were precipitated, purified, and assayed by quantitative PCR with the following primers: miR-221 forward 5′-GCTAAAGAGGGGGAGCAATC-3′, reverse 5′-CTGCTCTTTGAGGGAGGACAA-3′. The value of the ChIP samples was normalized to the input and was presented as a percentage of control.