ChIP Assay for PRC2 Targeting

Chromatin immunoprecipitation (ChIP) assay was performed to evaluate PRC2 targeting and H3K27me3 levels at specific promoters as described^{70 (link),71 (link)}. Briefly, minced hearts or NRVMs were fixed with 1% formaldehyde for 10 min at room temperature, and then quenched by 125 mM glycine. The samples were homogenized in lysis buffer containing 20 mM Tris-HCl (pH8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS, and 1× complete protease inhibitor tablet, and sonicated to generate chromatin samples with averaged fragment sizes of 200-1000 bp. After pre-cleared with Protein A sepharose beads, samples were incubated with anti-H3K27me3 antibody (Cell Signaling Technologies, MA, USA), anti-Ezh2 (Cell Signaling Technologies (#5246) for ChIP in mouse hearts ; Santa Cruz Biotechnology (#292275) for ChIP in NRVMs) or normal control IgG at 4°C over night on an inverse rotator. The antibody-chromatin complexes were then pelleted with BSA/Salmon sperm DNA blocked Protein A sepharose beads. After standard washes, the immunoprecipitated DNA was eluted and purified with PCR purification kit (Qiagen). RT-PCR was then performed using primers targeting the promoter regions of hypertrophy-related genes as listed in supplementary Table 5.