GSM8118935: ESC, SETb-HA, CHIP rep1; Mus musculus; ChIP-Seq
Following MEF depletion, 10 million  ESCs were harvested and crosslinked with 1% formaldehyde for 10 min and reaction was quenched with glycine.  DSG was added at a final concentration of 2 mM for 45 min at RT prior to addition of formaldehyde. Cells were lysed at 4°C for 10 minutes in lysis buffer 1 (50 mM HEPES-KOH 1 mM EDTA, 140 mM NaCl, 0.25% Triton X-100, 0.5% NP-40, 10% glycerol) 10 min in lysis buffer 2 (1 m mM Tris-HCl, 1 mM EDTA, 200 mM NaCl and 0.5 mM EGTA), and 30 min in lysis buffer 3 (10 mM Tris-HCl, 1 mM EDTA, 100 mM NaCl, 0.5mM EGTA and 0.1% Na-DOC). All lysis buffers were supplemented with protease inhibitors. Sonication was performed with Bioruptor® (Diagenode) for 27 cycles (30s on, 30s off), or until average DNA fragment size ranged from 300-500bp. Average sizes of libraries were determined by TapeStation (Agilent Technologies, USA). Samples were then incubated overnight at 4°C with Magna ChIP™ protein A beads (Sigma-Aldrich) coupled with 3 μg of HA (ab9110, Abcam) or SUZ12 (ab12073, Abcam) antibody. The beads were washed twice at 4°C with ice-cold RIPA buffer (10 mM Tris-HCl, 1mM EDTA, 140 mM NaCl, 1% Triton X-100, 0.1% SDS, and 0.1% Na-DOC) and once with RIPA-500 buffer (10 mMTris-HCl, 1 mM EDTA, 500 mM NaCl, 1% Triton X-100, 0.1% SDS, and 0.1% Na-DOC), LiCl wash buffer (10mM Tris-HCl, 1 mM EDTA, 250 mM LiCl, 0.5% NP-40, and 0.5% Na-DOC), TE buffer (10 mM Tris-HCl and 1 mM EDTA), and eluted from the beads in direct elution buffer (10 mM Tris-HCl, 5 mM EDTA, 300 mM NaCl, and 0.5% SDS). The supernatant was reverse-crosslinked with RNase and 1U Proteinase K per sample for at least 4 h at 65°C. Reverse-crosslinked DNA was purified with AMPure XP SPRI beads (Beckman). Quantification of DNA concentration was performed with Qubit fluorometric quantitation (Thermo Fisher Scientific). Library preparation was carried out as previously published (Blecher-Gonen et al., 2013). Libraries were amplified with a thermocycler using 12-13 cycles. Library DNA was purified with AMPure XP SPRI beads (Beckman). Quantification of DNA concentration was performed with Qubit fluorometric quantitation (Thermo Fisher Scientific).