title: GSM9365003: SUDHL5 cells, input for p300 ChIP-seq, 1 nM TCIP3 (0.1% DMSO) for 2 hrs; Homo sapiens; ChIP-Seq
library_construction_protocol: Cells were washed in PBS containing TCIP3 or DMSO and crosslinked for 11 min in CiA Fix Buffer (50 mM HEPES pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 100 mM NaCl) with addition of formaldehyde to a final concentration of 1%. The crosslinking reaction was quenched by glycine added at 0.125 M final concentration. Crosslinked cells were centrifuged at 1,000 x g for 5 min. Nuclei were prepared by 10 min incubation of resuspended pellet in CiA NP-Rinse 1 buffer (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% IPEGAL CA-630, 0.25% Triton X100) followed by wash in CiA NP-Rinse 2 buffer (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). The pellet was resuspended in CiA Covaris Shearing Buffer (0.1% SDS,1 mM EDTA pH 8.0, 10 mM Tris HCl pH 8.0) with protease inhibitors and sonicated for 20 min with a Covaris E220 sonicator (Peak Power 140, Duty Factor 5.0, Cycles/Burst 200). The distribution of fragments was confirmed with D1000 Tapestation or by agarose gel electrophoresis. 15 ug of chromatin per ChIP was used with anti-p300 antibodies (Bethyl Labs, cat#: A300-358A), with 12 ng Drosophila chromatin (53083, ActiveMotif) spiked in. 40 ug of chromatin per ChIP was used with anti-FLAG antibodies (Sigma, cat#: F1804) with 32 ng of Drosophila chromatin (53803, ActiveMotif) spiked in. After overnight incubation at 4 C in IP buffer (50 mM 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 (10 mM Tris pH 8, 0.25 M LiCl, 0.5% IPEGAL CA-630, 0.5% sodium deoxycholic acid salt (DOC), 1mM EDTA), and once with 10 mM Tris/1 mM EDTA buffer (TE) pH 8. IPs and inputs were reverse-crosslinked in TE/0.5% SDS/0.5 µg/µL proteinase K for 55 C /3 h then 65 C /18 h, then DNA was purified using a PCR cleanup spin column (Takara #74609). Paired-end sequencing libraries were constructed using an NEBNext Ultra II DNA kit (E7645S) and indexed (NEB E7335). Library size distributions were confirmed using an Agilent Bioanalyzer and High Sensitivity DNA reagents (Agilent 5067) and concentrations determined by qPCR.