GSM9267712: H3K4me3_ESC_rep1; Mus musculus; ChIP-Seq
Briefly, Bruce-4 cells were crosslinked in 1 % w/v formaldehyde at room temperature for 10 min and quenched with 125 mM glycine for 5 min at room temperature. Crosslinked cells were centrifuged at 1250rcf for 5 min at 4 ˚C and were washed twice with ice-cold PBS before snap-freezing. The cells were used for Rad21 ChIP (10 x 106), H3K27ac ChIP (15 x 106) and H3K4me3 ChIP (15 x 106). 5% of 1% fixed human MelI ESCs were added as a spike-in. For the Rad21 ChIP the cells were lysed in 1 ml cold cell lysis buffer (10 mM Tris pH 8.0, 10 mM NaCl and 0.2 % NP-40/Igepal (Sigma, I8896-50ML)) supplemented with protease inhibitors (Roche, 4693132001) for 20 min on ice. Nuclei were pelleted at 800g for 5 min, resuspended in 300 ul nuclear lysis buffer (50 mM Tris pH 8.0, 0.5% SDS and 10 mM EDTA) containing protease inhibitors and incubated on ice for 20 min as previously described.93  For the H3K27ac and H3K4me3 ChIP, cells were lysed in 300 ul lysis buffer (10 mM Tris pH 8, 1 mM EDTA and 0.5% SDS (Sigma, 05030-500ml-F) supplemented with protease inhibitors and incubated on ice for 20 min. The cells were sonicated in a Bioruptor device (30 s on/off; high setting) and spun down at the maximum speed for 15 min at 4 ˚C. The supernatants were precleared under rotation for 1h at 4 ˚C with protein G Dynabeads (Invitrogen, 10004D) pre-blocked with BSA-protein (Sigma, 9048-46-8). Next, the supernatants were diluted five times with dilution buffer (0.01% SDS, 1.1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8 and 167 mM NaCl) and incubated overnight with the respective antibody with rotation at 4 °C. Protein G Dynabeads preblocked with BSA protein were added the following day and incubated for 3-4h at 4 ˚C. The beads were immobilized on a magnet and washed twice with low-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl and 20 mM Tris pH 8), twice in high-salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl and 20 mM Tris pH 8), twice in LiCl buffer (0.25 M LiCl, 1% NP-40, 1% deoxycholic acid (sodium salt), 1 mM EDTA and 10 mM Tris pH 8) and once in TE buffer. DNA elution from the beads was done by incubating with 125 ul elution buffer (1% SDS and 10 mM NaHCO3) for 30 min at 65 ˚C 2000 rpm in a thermomixer. The supernatants and the inputs were reverse crosslinked by incubation overnight at 65 ˚C with X amount of proteinase K. After treatment with RNase A for 30 min at 37 ˚C, the DNA was purified using a kit for DNA cleaning and concentration (Zymo, zd4014). The immunoprecipitated DNA was used of library preparation using the KAPA Hyper prep kit (KAPA Biosystems, 7962347001).