GSM5513846: CDK9_RBBP5FKBP_0h; Mus musculus; ChIP-Seq
ES cells were cross-linked by the addition of 1% formaldehyde (Sigma-Aldrich, 252549-1L) in the dish for 10 minutes at RT before quenching with 0.125 M glycine. The fixed cells were washed with PBS and resuspended in SDS buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% SDS, 1x protease inhibitor cocktail from Roche). The resulting nuclei were spin down, resuspended in the immunoprecipitation buffer at 1 mL per 16 million cells (SDS buffer and Triton Dilution buffer (100 mM NaCl, 100 mM Tris-HCl pH 8.0, 5 mM EDTA, 5% Triton X-100) mixed in 2:1 ratio with the addition of 1x protease inhibitor cocktail from Roche) and processed on a Bioruptor Plus Sonicator (Diagenode) to achieve an average fragment length of 200-300 bps. Libraries were constructed using NEBNext Ultra II DNA Library prep kit (NEB, E7645L).