GSM4981376: LCL_H3K27me3_G2M; Homo sapiens; ChIP-Seq
Acetone-fixed cells were washed twice in cold PBS and resuspended in digestion buffer (0.32M sucrose, 50mM Tris/HCl (pH7.4), 4mM MgCl2, 1mM CaCl2, 5mM Na Butyrate, 0.1mM PMSF). Micrococcal nuclease was added to 25units/ml and chromatin digested at 37°C for 5 minutes. The reaction was stopped by the addition of EDTA to 5mM. Chromatin was pre-cleared by incubation with protein A sepharose beads and incubated overnight with antibodies to H3K9ac (in house, R607), H3K4me3 (in house, R612) and H3K27me3 (Millipore, 07-449). Antibody-bound material was isolated on Protein A-Sepharose beads (Invitrogen, UK) and DNA from antibody-bound and input chromatin was purified by PCR purification kit (Qiagen). Sequencing libraries were prepared from 100ng DNA per sample using the NEBNext Library preparation kit for Illumina (NEB).  Samples were barcoded using the system described by Bronner et al. (2014).  Fragments were PCR amplified with 12–18 cycles using adaptor specific primers (Illumina); fragments ranging between 300 and 500 bp in size were gel-purified before cluster generation and sequencing.