Megna chip a g chromatin immunoprecipitation chip kits

H3K27me2 in vehicle-, IL-1β-, EZH2 cDNA-, EZH2 RNAi-, and UNC1999-treated chondrocyte cultures (5 × 10⁶ cells) was immunoprecipitated using H3K27me2 antibody, IgG, and Megna ChIP™ A/G Chromatin Immunoprecipitation (ChIP) kits (Millipore, Temecula, CA), according to the manufacturer’s instructions. To isolate DNA, the immunoprecipitates were subjected to sonication, elution, Proteinase K digestion, and enrichment with column elution. For PCR assessment, aliquots of DNA specimens were pipetted with Cy3-conjuated probes (forward: 5′-ACGACAGATTGAAGGCCTGGG-3′; reverse: 5′-GGTGCTCTTCCCCAATCAT-3′) (Applied Biosystems) for the −688 to −427 bp region proximal to the transcription start site of the miR-128a promoter (Ensembl:ENSG00000207654). The positive control GADPH promoter was detected using another Cy3-conjugated probe (forward: 5′-TACTAGCGGTTTTAC GGGCG-3′; reverse: 5′-TCGAACAGGAGGAGCAGAGAGCGA-3′). To test the amplification efficiency, standard curves of Ct values vs. serial fivefold dilutions of DNA were plotted and H3K27me2 enrichment to the miR-128a promoter was expressed as % input DNA.

H3H27ac immunoprecipitates in 10⁷ cells were isolated using H3K27ac antibody and IgG together with Megna ChIP™ A/G Chromatin Immunoprecipitation (ChIP) kits (Millipore, Temecula, CA, USA). The immunocomplexes were further processed with proteinase K and sonication to extract DNA. In all, 1 ng DNA specimen was subjected to PCR analysis using Cy3-conjuated probes (Supplementary Table 1) for the -1991~+ 8 bp region proximal to CXCL12 promoter (ENSMUSG00000061353), respectively. PCR assay of GADPH promoter (ENSMUSG00000207654) was designated as positive controls. H3K27ac enrichment to the promoter of interest was expressed as % input DNA.