P-GSE42560-12 - P-GSE42560-12
nucleic acid extraction protocol
ChIP of modified histones was done as described (Sørensen et al 2010 Mol Biol Cell) using antibodies to H3K9me3 (Diagenode, pAb-056-050), H3K27me3 (Millipore, 07-449) and H3K4me3 (Abcam, Ab8580). In short, cells were cross-linked for 8 min with 1% formaldehyde and sonicated to ~400 bp fragments using a Bioruptor (Diagenode). Chromatin (100 µl at 0.5 A260 U/µl) was incubated with 2.4 μg antibody coupled to Dynabeads Protein A (Invitrogen). ChIP material was washed, RNAse-treated, purified by phenol-chloroform isoamylalcohol extraction, amplified using the WGA4 kit (Sigma-Aldrich), cleaned up and eluted in MilliQ water. For ChIP of LMNA, cells were cross-linked as above and chromatin prepared by lysis (5 min on ice) and sonication (total of 45 min in a Bioruptor) in the following lysis buffer: 10 mM Tris-HCl, pH 7.5, 10 mM KCl, 2 mM EDTA, 1% Triton X-100, protease inhibitors. Chromatin (100 µl at 2 A260 U/µl) was incubated overnight at 4oC with 2.4 μg anti-lamin A/C antibody (Santa Cruz, sc-7292) coupled to Dynabeads Protein A (Invitrogen). ChIP material was washed in lysis buffer and processed as for histone ChIPs. MeDIP was performed as described (Sørensen and Collas, 2009. Meth Mol Biol 567, 249-262). In short, genomic DNA was purified and fragmented by probe sonication to 300-500 base pair fragments. Methylated fragments were immunoprecipitated using anti-5 mC antibodies (Diagenode). Immunoprecipitated (MeDIP) and input DNA was amplified by 14 PCR cycles using the WGA2 kit (Sigma-Aldrich) and cleaned up using the MinElute PCR purification kit (Qiagen). Input and MeDIP DNA were labeled with Cy3 and Cy5 respectively and hybridized on the same promoter arrays as those used for ChIP-chip.