title: GSM7421832: HEK293 T-Rex Flp-In cells, BRD4-NUT, input; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were grown in 15cm dishes to confluency and 2-3 plates were used for each experiment. After doxycycline treatments, cells were washed once with 20mL of PBS. Next, the cells were treated with 1% methanol-free formaldehyde diluted in PBS – 10mL per plate for 8 minutes, with gentle agitation. Formaldehyde was then quenched with 500μL of 2.5M glycine for another 8 minutes, with gentle agitation. After quenching, cells were scraped off the plates, transferred to 50mL conical tubes and centrifuged at 500xg at 4oC for 5 minutes. The supernatant was removed, and the cell pellet was washed with 5mL of cold PBS per plate. Following the wash, cells were again centrifuged at 500xg at 4oC for 5 minutes and then, the supernatant was removed. Pellets were finally snap-frozen in liquid nitrogen and stored at –80oC. Cell pellets were thawed and resuspended in LB1 buffer (50mM Hepes-KOH pH 7.9, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% NP40, 1% TritonX-100 and 1x Complete, EDTA-free protease inhibitor cocktail) to obtain ~1x107 cells/mL suspension and they were then incubated on a rotator at 4oC for 20 minutes, for lysis. Lysis efficiency of at least 60% was determined using trypan blue. Next, the suspension was centrifuged at 1,350xg at 4oC for 5 minutes. The supernatant was removed, and the pellet was resuspended in LB2 buffer (10mM Tris pH 8.0, 200mM NaCl, 1mM EDTA, 0.5mM EGTA and 1x Complete, EDTA-free protease inhibitor cocktail) to again obtain ~1x107 cells/mL suspension and they were then incubated on a rotator at 4oC for 5 minutes. Next, the suspension was centrifuged at 1,350xg at 4oC for 5 minutes. The supernatant was removed, and the pellet was resuspended in LB3 buffer (10mM Tris pH 8.0, 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% sodium deoxycholate, 0.5% SDS, 1% TritonX-100 and 1x Complete, EDTA-free protease inhibitor cocktail) to obtain ~1x107 cells/mL suspension, which was then passed through a 27G needle 3 times, to completely homogenize the pellet. The suspension was transferred to a Covaris millitube with AFA fiber and sonicated using Covaris M220 sonicator (average incident power 7.5 Watts, peak incident power 75 Watts, duty factor 10%, cycles: 200 count, duration: 18 minutes, minimum temperature: 5oC, temperature setpoint: 7oC, maximum temperature: 9oC). After sonication, samples were centrifuged at 15,000xg at 4oC for 10 minutes, soluble supernatant was transferred to a new tube, snap-frozen in liquid nitrogen and stored at –80oC. 10μL of each sample were incubated overnight at 65oC and treated with Proteinase K and RNAse A and DNA was purified to test the sonication efficiency. Protein G-conjugated Dynabeads (75uL of suspension per IP) were washed with 1mL of blocking buffer (0.5% BSA in PBS) 3 times and collected on a magnet stand each time. Beads were resuspended in 500μL blocking buffer and mixed with 7.5μg antibody (NUT monoclonal rabbit antibody or H3K27Ac monoclonal rabbit antibody) and incubated on a rotator at 4oC overnight. Next day, the beads were washed 3 times with the same blocking buffer to remove unbound antibody and then they were resuspended in 75μL of the blocking buffer. The resuspended beads bound to antibody were mixed with chromatin extract and mixed on a rotator at 4oC overnight. After that, the beads were washed 3 times in 1mL washing buffer 1 (50mM Hepes pH 7.0, 100mM LiCl, 1mM EDTA, 1% NP40, 0.7% sodium deoxycholate and 1x Complete, EDTA-free protease inhibitor cocktail). For the third wash, beads were incubated on a rotator at 4oC for 10 minutes. The washes were repeated with washing buffer 2 (20mM Tris pH 8.0, 350mM NaCl, 1% TritonX-100, 0.1% SDS, 2mM EDTA). Next, the beads were washed with 1mL TE buffer with 50mM NaCl and centrifuged shortly at 300xg and then placed on the magnet stand, to remove residual buffer. Then, 200μL of the elution buffer was added to the beads (50mM Tris pH 8.0, 10mM EDTA and 1% SDS) and the beads were incubated at 65oC for 30 min. with gentle agitation. The beads were next centrifuged at 300xg and placed on the magnet stand. The 200μL elution was then transferred to a new tube. The elution was incubated at 65oC overnight to reverse crosslinks. Next, samples were treated with RNase A for 1h at 37oC and then with Proteinase K for 2h at 55oC. DNA was then purified from these samples using the Qiagen PCR purification kit.