title: GSM5578024: CM_P1_cKO_rep2_H3K79me2; Mus musculus; ChIP-Seq
library_construction_protocol: Cardiomyocytes were isolated using collagenase enzymatic reaction and FACS sorted based on the tdTomato fluorescence driven by the XMlc2-Cre. Purified Cardiomyocytes were cross-linked using 1% formaldehyde/PBS for 10 minutes at room temperature, the reactions quenched by adding 2.625 M glycine to 125 mM final 20% BSA to 0.5% final and cells pelleted by centrifugation for 5 minutes at 1,000 g, 4°C. Cells were washed twice with ice-cold 0.5% BSA/PBS and cell pellets were snap-frozen in liquid nitrogen and stored at −80°C. Fixed cells were thawed on ice, resuspended in 500 µl ice-cold buffer L2 (0.5% Empigen BB, 1% SDS, 50 mM Tris/HCl pH 7.5, 1 mM EDTA, 1 × protease inhibitor cocktail) and chromatin was sheared to an average DNA size of 300–500 bp by administering 7 pulses of 10 s duration at 13 W power output with 30 s pause on wet ice using a Misonix 3000 sonicator. The lysate was diluted 2.5-fold with ice-cold L2 dilution buffer (20 mM Tris/HCl pH 7.4@20°C, 100 mM NaCl, 0.5% Triton X-100, 2 mM EDTA, 1 × protease inhibitor cocktail), and one percent of the lysate was kept as ChIP input. For each immunoprecipitation, aliquots of diluted lysate equivalent to 150,000 to 1 million cells, 20 μl of Dynabeads Protein A (for rabbit polyclonal antibodies) and 2 μg anti H3K79me2 antibody (Abcam #ab3594) were combined and rotated overnight at 8 RPM and 4°C. The following day, beads were collected on a magnet and washed three times each with wash buffer I (10 mM Tris/HCl pH 7.5, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 2 mM EDTA), wash buffer III (10 mM Tris/HCl pH 7.5, 250 mM LiCl, 1% IGEPAL CA-630, 0.7% Deoxycholate, 1 mM EDTA) and twice with ice-cold TET (10 mM Tris/HCl pH7.5, 1 mM EDTA, 0.2% Tween-20). Libraries were prepared directly on the antibody/chromatin-bound beads: after the last TET wash, beads were suspended in 25 μl TT (10 mM Tris/HCl pH7.5, 0.05% Tween-20). Libraries were prepared using NEBNext Ultra II reagents according to the manufacturer's protocol but with reagent volumes reduced by half. DNA was eluted and crosslinks reversed by adding 4 μl 10% SDS, 4.5 μl 5 M NaCl, 3 μl EDTA, 1 μl proteinase K (20 mg/ml), 20 μl water, incubating for 1 h at 55°C, then overnight at 65°C. DNA was cleaned up by adding 2 μl SpeedBeads 3 EDAC in 61 μl of 20% PEG 8000/1.5 M NaCl, mixing and incubating for 10 minutes at room temperature. SpeedBeads were collected on a magnet, washed twice by adding 150 μl 80% EtOH for 30 s each, collecting beads and aspirating the supernatant. After air-drying the SpeedBeads, DNA was eluted in 25 μl TT and the DNA contained in the eluate was amplified for 12 cycles in 50 μl PCR reactions using NEBNext High-Fidelity 2X PCR Master Mix or NEBNext Ultra II PCR master mix and 0.5 μM each of primers Solexa 1GA and Solexa 1GB. Libraries were cleaned up as above by adding 36.5 μl 20% PEG 8000/2.5 M NaCl and 2 μl Speedbeads, two washes with 150 μl 80% EtOH for 30 s each, air-drying beads and eluting the DNA into 20 μl TT. ChIP library size distributions were estimated following 2% agarose/TBE gel electrophoresis of 2 μl library, and library DNA amounts measured using a Qubit HS dsDNA kit on a Qubit fluorometer. ChIP input material (1 percent of sheared DNA) was treated with RNase for 15 min at 37°C in EB buffer (10 mM Tris pH 8, 0.5% SDS, 5 mM EDTA, 280 mM NaCl), then digested with Proteinase K for 1 h at 55°C and crosslinks reversed at 65°C for 30 min to overnight. DNA was cleaned up with 2 μl SpeedBeads 3 EDAC in 61 μl of 20% PEG 8000/1.5 M NaCl and washed with 80% ethanol as described above. DNA was eluted from the magnetic beads with 25 μl of TT and library prep and amplification were performed as described for ChIP samples. Libraries were sequenced single-end for 75 cycles (SE75) to a depth of 20-25 million reads on an Illumina NextSeq 500 instrument. .