GSM4849110: ChIP_H3K4me3_Ery_iPSC_Vs_Clone2_rep1; Homo sapiens; ChIP-Seq
isolation protocol: Cells were fixed at 2.5x10^6 cells/ml of 10% FCS RPMI media (Gibco) with 1% formaldehyde (SIGMA) for 10min at RT. Fixed cells were lysed at 5x10^7 cells/ml of cell lysis buffer (5mM PIPES (Gibco), 85mM KCl, 0.5% NP-40 (SIGMA)) + proteinase inhibitor (PI) (ROCHE) on ice for 20min. The lysed fixed nuclei were lysed in nuclear lysis buffer (50mM Tris-HCl, 10mM EDTA (both from Life Technologies) 1% SDS (SIGMA)) +PI at a concentration of 1x10^8 cells/ml. The nuclear lysis was sonicated using the Covaris S220 Focused-ultrasonicator in 130μl microTUBEs (Covaris). The sonicated lysate was spun at max speed at 10°C to remove insoluble material. The clear fixed chromatin was diluted 1 in 10 with RIPA buffer w/o SDS +PI (10mM Tris-HCl, 1mM EDTA, 0.5mM EGTA (SIGMA), 1% Triton X-100 (SIGMA), 0.1% Na Deoxycholate (SIGMA), 140mM NaCl). The diluted chromatin was precleared twice with 10µl of a 1:1 mix of Protein A and Protein G Dynabeads (Invitrogen) by incubating at 4°C for 30mins and discarding the beads. The pre-cleared chromatin was incubated with the appropriate amount of antibody coupled to a 100µl 1:1 mix of Protein A and Protein G Dynabeads at 4°C overnight. The antibodies used are: RNA Polymerase II N-20 antibody SC-899 (Santa Cruz Biotechnology) 6μg per 1ml of chromatin from 1x10^7 cells; Monoclonal anti-acetyl-Histone H3 (Lys27) antibody 17-683 (Merck Millipore) 2μg per 1ml of chromatin from 1x10^7 cells; Polyclonal anti-trimethyl-Histone H3 (Lys4) antibody 07-473 (Merck Millipore) 1μg per 1ml of chromatin from 1x10^7 cells; Polyclonal anti-monomethyl-Histone H3 (Lys4) antibody ab195391 (Abcam) 4μg per 1ml of chromatin from 1x10^7 cells; Polyclonal anti-GATA1 antibody ab11852 (Abcam) 8μg per 1ml of chromatin from 1x10^7 cells; Policlonal anti-CTCF antibody 07-729 (Merck Millipore) 10μl serum per 1ml of chromatin from 1x10^7 cells. Polyclonal anti-KLF1 antibody was kindly provided by the Perkins Laboratory, Translational Research Institute, Brisbane, Australia (used 30μl serum per 1ml of chromatin from 1x10^7 cells). Chromatin bound beads were washed twice with RIPA (10mM Tris-HCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Na Deoxycholate, 140mM NaCl) +PI, then twice with high salt RIPA (same as RIPA but with 500mM NaCl) +PI, once with LiCl RIPA (same as RIPA but with 250mM LiCl instead of NaCl) +PI and twice with TE buffer. Chromatin was eluted off beads using 20mM Tris-HCl, 5mM EDTA, 50mM NaCl, 1% SDS, 130µg/ml RNase A, 65µg/ml Proteinase K. Eluted chromatin and Input samples were incubated at 65°C overnight to de-crosslink DNA from protein. DNA was purified by phenol-chloroform extraction and ethanol precipitation with NaOAc, and 1 µl GlycoBlue (Invitrogen). ChIP enrichment was determined by RT-qPCR NEBNext Ultra II DNA Library Prep kit for Illumina (NEB).