Anti h3k27me3 antibody

When osteoclast precursors were treated for 72 h, they were fixed using 1% formaldehyde, washed and collected by centrifugation (1,000g for 5 min at 4 °C). The pellet was resuspended in RIPA buffer (50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.5% sodium deoxycholate, 1% SDS and 1% protease inhibitors), homogenized, incubated on ice for 10 min and sonicated. The samples were centrifuged (16,000g for 10 min at 4 °C) and from the supernatant, shared chromatin was used as input and incubated with an anti-H3K27Me3 antibody (9733, Cell Signaling Technology). Rabbit IgG (2729, Cell Signaling Technology) was used as the isotype control. After pull-down using Pierce Protein A/G Magnetic Beads (Thermo Fisher Scientific), followed by RNA and protein digestion, DNA was purified using Agencourt AMPure XP (Beckman Coulter) according to the manufacturer’s instructions. RT–qPCR was performed using SYBR GreenER qPCR SuperMix Universal (Thermo Fisher Scientific) and specific primers to detect H3K27Me3 enrichment in the transcription start site of Nfatc1 (set 1: Fw 5′-CAGCGACATGAAAGGAACAATC-3′, Rev 5′-GGACACCTGGCTCATCTTTAG-3′; set 2: Fw 5′-GGCAGAACTCTTGTCTGGATAC-3′, Rev 5′-GCCTTAGCTGCTTCTCACTAAA-3′; set 3: Fw 5′-GGTCAAGTTATCCCTGCTGAA-3′, Rev 5′-ACAATGACATGACCCAGACC-3′).

ChIP assay was carried out according to the instruction provided with the ChIP assay kit (SimpleChIP® Enzymatic Chromatin IP Kit, Cat# 9003, Cell Signaling Technology) according to previous published method with some modifications^{44 (link)}. In brief, the cells were first crosslinked with 1% formaldehyde for 10 min at RT and then quenched with 0.125 mol/L glycine. Afterwards, chromatin was sonicated in the lysis buffer to 100–300 bp DNA fragments. Immunoprecipitation was performed using an anti-BMI1 antibody (Cell Signaling Technology-#6964), anti-H3K27me3 antibody (Cell Signaling Technology-#9733) or a normal Rabbit IgG (Cell Signaling Technology-#2729). The immunoprecipitated DNA was finally harvested from the beads and analyzed by PCR. The promoter primer sequences of the PRE sites for miR-218-1-3p promoter were given as follows: PRE site 1 forward: 5’-CAAAGGAAGGGTTAAACGGAAGG-3’, reverse: 5’-CGCTTACAGACACACACGTG-3’; PRE site 2 forward: 5’- CTAGAAGGAGAAGGACTACC-3’, reverse: 5’-TTCCCAGGCTTTGAATTCCG- 3’. For the EZH2 inhibitor experiments, PLC^BMI1 cells were first treated with or without 15 nM PF-067263004 for 24 h before conducting ChIP assays as described above.

The assay was performed using an EZ-Zyme Chromatin Prep Kit (Millipore), according to the manufacturer’s protocol. Anti-H3K27me3′ antibody (Cat# 9783, Cell Signal Technology) was used in the immunoprecipitation reaction with H3K27me3. The pulled DNA underwent purification and was analyzed by RT-qPCR with primers specific to the predicted binding sites on the promoter site. Immunoprecipitated DNA and whole-cell extracted DNA were also treated for reverse crosslinking using the Zymoclean PCR purification kit (Zymo). The ChIP-PCR primers are listed in Supplementary Table S1.

ChIP was performed as previously described [46 (link)] on chromatin prepared from dorsal midbrains of E11.5 NMRI embryos. A rabbit mab anti-H3K27me3 antibody (Cell Signaling Technology, #9733, 1:250) was used. Purified DNA (1 μL) was used as input for the quantitative real-time PCR and the reaction was carried out using LightCycler® SYBR Green I master mix (Roche, 4887352001), run on a LightCycler® 480 System (Roche). Primers were designed to amplify genomic DNA from a region flanking the transcriptional starting site ±500 bp and is devoid of local CpG islands. Primers used are listed in Table S4 (Additional file 1: Table S4). Obtained data were analyzed by the ∆Ct method and normalized to ChIP input. Also, intragenic region Int1 (chr5: 79227331–79229070) is unmethylated and served as negative control.