mouse adult cardiac myocyte nuclei rep3:H3K27me3
ChIP-seq was performed as previously described with modifications (Bonn, S. et al. Nature protocols 7, 978-994, 2012). Lysis buffers for nuclei preparation were supplemented with 10 mM sodium butyrate (Sigma, Munich, Germany). MACS purified nuclei were fixed with 1% freshly prepared PFA (Paraformaldehyde, Sigma, Munich, Germany) for 2 min at room temperature. Fixation was stopped by adding glycine to a final concentration of 125 mM. Nuclei were washed three times with PBS, were subsequently lysed in SDS lysis buffer (50 mM Tris-HCl (pH 8.0), 10 mM EDTA, 1% SDS) supplemented with protease inhibitors (Roche Complete Protease Inhibitor Cocktail, Roche, Mannheim, Germany) and chromatin was sheared using a Bioruptor (Diagenode, Liege, Belgium) with 30 high-energy cycles (30s on/30s off) to an average size of 200-400 bp. Lysates were diluted 1 in 10 with dilution buffer (22 mM Tris-HCl (pH 8.0), 165 mM NaCl, 2.2 mM EDTA, 1.1% Triton X-100) and were precleared with 0.1% BSA for 2h at 4°C. Antibodies were preincubated with Protein A Dynabeads (Invitrogen, Karlsruhe, Germany) for 2h at 4°C. For immunoprecipitation either 5 µg of chromatin was incubated with antibodies directed against H3K4me3 (2 µg, pAb-003-050, Diagenode) and H3K27ac (2 µg, ab4729, Abcam) over night at 4°C or 2.5 µg of chromatin was incubated with an anti-H3K4me1 antibody (1 µg, ab8895, Abcam). Immunocomplexes were washed for 10 min each with buffer 1 (20 mM Tris-HCl (pH 8.0), 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), buffer 2 (20 mM Tris-HCl (pH 8.0), 500 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS), buffer 3 (20 mM Tris-HCl (pH 8.0), 250 mM LiCl, 2 mM EDTA, 1% IGEPAL CA-630, 1% sodium deoxycholate) and three times with TE buffer (20 mM Tris-HCl (pH 8.0), 2 mM EDTA) at 4°C. Elution from beads was carried out by shaking in elution buffer (100 mM NaHCO3, 1% SDS) at 1400rpm at room temperature for 1h. DNA was treated with 50 ng/µl RNAse A (Sigma, Munich, Germany) for 30min at 37°C and crosslinks were reversed by adding 0.5 µg/µl Proteinase K (AppliChem, Darmstadt, Germany) and incubation at 65°C for 5h. DNA was purified using Phenol/Chloroform followed by ethanol precipitation -20°C over night. DNA was quantified using Quant-iT PicoGreen dsDNA Reagent (Invitrogen, Karlsruhe, Germany). Sequencing libraries were prepared from 5ng DNA with the NEBNext ChIP-seq Library Prep Master Mix Set for Illumina (NEB, Frankfurt, Germany) according to manufacturer instructions with 15 PCR cycles and sequenced on a HiSeq 2000 (50 bp, paired-end, Illumina, San Diego, USA).