GSM4973587: ChIP_A375_WT_H3K36me2; Homo sapiens; ChIP-Seq
Culture media was removed and plates were washed once with PBS and then fixed for 10 minutes with 1% formaldehyde (Thermo Fisher Scientific) in PBS at room temperature and reaction was then quenched by adding glycine (Thermo Fisher Scientific) to 0.125M. After fixation, cells were washed once with cold PBS and then scraped into supernatant using a rubber policeman, pelleted for 5 minutes at 400xG at 4°C. Cells were transferred to Eppendorf DNA Lobind tubes and pelleted at 700xG for 5 minutes at 4°C, snap-frozen in liquid nitrogen and stored at -80°C until ready for ChIP-seq protocol preparation. Samples were sonicated using a probe sonicator in 500 µl lysis buffer (10 mM Tris/HCl pH 7.5, 100 mM NaCl, 1 mM EDTA, 0.5mM EGTA, 0.1% deoxycholate, 0.5% sarkozyl, 1 × protease inhibitor cocktail). After sonication, 10% Triton X-100 was added to 1% final concentration and lysates were spun at full speed for 10 minutes. 1% was taken as input DNA, and immunoprecipitation was carried out overnight with 20 µl Protein A Dynabeads (Invitrogen) and 2 µg specific antibodies. Beads were washed three times each with wash buffer I (20mM Tris/HCl, 150mM NaCl, 0.1% SDS, 1% Triton X-100, 2mM EDTA), wash buffer II (10mM Tris/HCl, 250mM LiCl, 1% IGEPAL CA-630, 0.7% Na-deoxycholate, 1mM EDTA), TE 0.2% Triton X-100 and TE 50mM NaCl and subsequently resuspended 25 µl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20 and sequencing libraries were prepared on the Dynabeads as described below. ChIP libraries were prepared while bound to Dynabeads using NEBNext Ultra II Library preparation kit (NEB) as previously described (Heinz et al., 2018). DNA was polished, polyA-tailed and ligated after which dual UDI (IDT) or single (Bioo Scientific) barcodes were ligated to it. Libraries were eluted and crosslinks reversed by adding to the 46.5 µl NEB reaction 16 µl water, 4 µl 10% SDS, 4.5 µl 5M NaCl, 3 µl 0.5 M EDTA, 4 µl 0.2M EGTA, 1 µl RNAse (10 mg/ml) and 1 µl 20 mg/ml proteinase K, followed by incubation at 55C for 1 hour and 75C for 30 minutes in a thermal cycler. Dynabeads were removed from the library using a magnet and libraries were cleaned up by adding 2 µl SpeedBeads 3 EDAC (Thermo) in 124 µl 20% PEG 8000/1.5 M NaCl, mixing well, then incubating at room temperature for 10 minutes. SpeedBeads were collected on a magnet and washed two times with 150 µl 80% ethanol for 30 seconds. Beads were collected and ethanol removed following each wash. After the second ethanol wash, beads were air dried and DNA eluted in 12.25 µl 10 mM Tris/HCl pH 8.0 and 0.05% Tween-20. DNA was amplified by PCR for 14 cycles in a 25 µl reaction volume using NEBNext Ultra II PCR master mix and 0.5 µM each Solexa 1GA and Solexa 1GB primers. Libraries were size selected using TBE gels for 200 – 500 bp and DNA eluted using gel diffusion buffer (500 mM ammonium acetate, pH 8.0, 0.1% SDS, 1 mM EDTA, 10 mM magnesium acetate) and purified using ChIP DNA Clean & Concentrator (Zymo Research).