GSM7845499: SUDHL5, Pol II, ChIP-seq, 30 nM CDK-TCIP1 treated, 4hr, rep2; Homo sapiens; ChIP-Seq
25-30 million cells were treated with CDK-TCIP1 or DMSO for indicated timepoints. Cells were washed in PBS containing CDK-TCIP1 and crosslinked for 12 min in CiA Fix Buffer (50mM HEPES pH 8.0, 1mM EDTA, 0.5mM EGTA, 100mM NaCl) with addition of formaldehyde to a final concentration of 1%. The crosslinking reaction was quenched by glycine added at 0.125M final concentration. Crosslinked cells were centrifuged at 1,000 x g for 5min. Nuclei were prepared by 10 min incubation of resuspended pellet in CiA NP-Rinse 1 buffer (50mM HEPES pH 8.0, 140mM NaCl, 1mM EDTA, 10% glycerol, 0.5% IPEGAL CA-630, 0.25% Triton X100) followed by wash in CiA NP-Rinse 2 buffer (10mM Tris pH 8.0, 1mM EDTA, 0.5mM EGTA, 200mM NaCl). The pellet was resuspended in CiA Covaris Shearing Buffer (0.1% SDS,1mM EDTA pH 8.0, 10mM Tris HCl pH 8.0) with 1000x protease inhibitors (Roche) and sonicated for 40 min with Covaris E220 sonicator (Peak Power 140, Duty Factor 5.0, Cycles/Burst 200). The distribution of fragments was confirmed with D1000 Tapestation. 50 µg of chromatin per ChIP was used with anti-CDK9 antibodies (Cell Signaling CST2361S, abcam ab239364), with 40ng Drosophila chromatin (53083, ActiveMotif) spiked in. 50µg of chromatin was used with anti-Pol II ser 2 phos (2 µg, Abcam ab5095) and anti-Pol II ser 5 phos (2 µg, Abcam ab5131) antibodies, and anti-Pol II (5 µg, Santa Cruz 8WG16) antibodies. After overnight incubation at 4OC in IP buffer (50mM HEPES pH 7.5, 300mM NaCl, 1mM EDTA, 1% Triton X100, 0.1% sodium deoxycholic acid salt (DOC), 0.1% SDS), IPs were washed twice with IP buffer, once with DOC buffer (10mM Tris pH 8, 0.25M LiCl, 0.5% IPEGAL CA-630, 0.5% sodium deoxycholic acid salt (DOC), 1mM EDTA), and once with 10mM Tris/1mM EDTA buffer (TE) pH 8. IPs and inputs were reverse-crosslinked in TE/0.5% SDS/0.5µg/µL proteinase K for 55°C/3 hrs then 65OC/18 hrs, then DNA was purified using a PCR cleanup spin column (Takara #74609). The sequencing library preparation was performed using NEBNext Ultra II DNA kit (#E7645S).