title: mouse adult cardiac myocyte nuclei: H3K36me3
design: FACS sorted cardiomyocyte nuclei were fixed with 1 % PFA (paraformaldehyde, Sigma) for 2 min at room temperature. Fixation was quenched by adding glycine to a final concentration of 125 mM. After washing with PBS for three times, nuclei were lysed in SDS buffer (50 mM Tris-HCl pH 8.0, 10 mM EDTA, 1 % SDS) supplemented with protease inhibitors (cOmplete Protease Inhibitor Cocktail, Roche). Chromatin was sonicated using a Bioruptor® (Diagenode) with 30 cycles (30 s on/30 s off; high energy) to an average fragment size of 200 – 400 bp. 200 µl  Protein A Dynabeads (Life technologies) were washed with TE buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA) two times. Following the second wash step, beads were resuspended in 1 ml TE buffer containing 1 mg/ml tRNA (Sigma) and 1 mg/ml BSA (Roth). After incubation for 2 h at 4 °C, beads were washed with TE buffer three times and finally resuspended in 200 µl TE. Chromatin concentration was adjusted to 50 ng/µl using SDS buffer. Next, chromatin was diluted 1:10 with dilution buffer (50 mM HEPES-KOH pH 7.5, 500 mM NaCl, 1 mM EDTA, 1 % Triton X-100, 0.1 % sodium deoxycholate, 0.1 % SDS, protease inhibitors) and precleared with 10 µl blocked beads for 1 h at 4 °C. 50 µl of preblocked chromatin served as input. For immunoprecipitation 2.5 µg of chromatin was incubated with an anti-H3K36me3 antibody (1 µg, ab9050, Abcam) overnight. The next day, 15 µl blocked beads were added and samples were incubated for 3 h at 4 °C. Immunocomplexes were washed for 5 min each with dilution buffer for two times, with wash buffer (10 mM Tris-HCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5 % IGEPAL CA-630, 0.5 % sodium deoxycholate) and finally with TE buffer (20 mM Tris-HCl pH 8.0, 2 mM EDTA) at 4 °C. Following magnetic separation, beads were resuspended in 160 µl elution buffer (100 mM NaHCO3, 1 % SDS) and immunocomplexes were eluted by incubation at room temperature for 15 min. This step was repeated with another 160 µl of elution buffer. The eluates were combined and treated with 20 ng/µl RNAse A (Sigma) for 30 min at 37 °C. Crosslinks were reversed by adding 0.5 µg/µl proteinase K (AppliChem) and incubation at 55 °C for 2.5 h. After this initial incubation, temperature was raised to 65 °C and incubated overnight. DNA was purified using phenol/chloroform followed by ethanol precipitation at 20 °C overnight. DNA was quantified using Quant-iT™ PicoGreen® dsDNA Reagent (Life technologies) and 5 ng were used per library. NEBNext® ChIP-seq Library Prep Master Mix Set for Illumina (NEB) was used for library preparation (15 PCR cycles). Libraries were sequenced on a HiSeq™ 2500 (50 bp, paired-end, Illumina).