Anti h3k27me3 antibody

ChIP assay was performed using EpiQuik™ Chromatin Immunoprecipitation Kit (P-2002; Epigentek, Farmingdale, NY, USA) according to the manufacturer’s instructions. NSCLC cells were cross-linked and sonicated as previously described [24 (link)]. The sheared chromatin fragments (approximately 200–500 bp) were subjected to immunoprecipitation with ChIP-grade antibodies as follows: anti-SET1AD antibody (A300-289A; Bethyl Lab), anti-H3K4me3 antibody (ab8580; Abcam), anti-β-catenin (51067–2-AP; Proteintech), anti-WDR5 antibody (#13105; Cell Signaling Technology), anti-H3K27me3 antibody (39,157; Active Motif), anti-H3K27ac antibody (39,134; Active Motif) and normal Rabbit IgG (#2729; Cell Signaling Technology). The primers for ChIP-PCR were listed in Additional file 1: Table S1.

Day 6 uteri from the control and Ezh2 uKO were used. Uterine tissues surrounding the embryos were longitudinally opened at the mesometrial side and kept at −80 °C until use. Endometrial tissues were disrupted with 35 strokes in a Dounce homogenizer on ice, with a loose-fitting pestle in PBS-containing protease inhibitor cocktail and phosphatase inhibitors 2 and 3 (Sigma, St. Louis, MO, USA). After centrifugation with 1000g for 5 min, pellets were received in the nuclei EZ lysis buffer (Sigma) to isolate the nuclei. Native ChIP was then performed as previously described [63 (link)], with some modifications. For the fragmentation, chromatins were treated by 20 U/μl MNase at 37 °C for 5 min. Input DNA was analyzed with a 2100 Bioanalyzer system using High-Sensitivity DNA Reagent kit (Agilent, Santa Clara, CA, USA) to confirm DNA fragmentations at approximately 200–300 bps. Chromatin immunoprecipitation was performed using Magna ChIP G-Chromatin Immunoprecipitation Kit (Millipore, Burlington, MA, USA) following the manufacturer’s protocol. Anti-H3K27me3 antibody (39155, Active motif, Carlsbad, CA, USA) or anti-rabbit IgG (2729, Cell Signaling Technology) was used to precipitate the target chromatins. Chromatin-immunoprecipitated DNAs were purified by extracting phenol–chloroforms.