GSM9292748: p300KO_Triptolite_0J_exp_tript_rep1; Homo sapiens; ChIP-Seq
Cells were cross-linked with ChIP Cross-link Gold (Diagenode, #C01019027) following manufacturer instructions. After incubation at room temperature for 30 min, the cells were fixed with a 1% formaldehyde solution (Thermo Fisher Scientific, #28908) for 10 min at room temperature. To stop the fixation, 125 mM glycine was added and incubated for 5 min. The cells were then washed twice with PBS and harvested on ice using cell scrapers. The harvested cells were resuspended in ChIP lysis buffer supplemented with protease inhibitors (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1% Triton X-100, 0.25% SDS) and sonicated on a Bioruptor Pico for 4 rounds of 10 cycles each at 4 °C with a short vortex between rounds to generate DNA fragments <500 bp. The sonicated cells were centrifuged, and the supernatants were transferred to new tubes and diluted with one volume of IP Dilution buffer (1% Triton X-100, 150 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 20 mM Tris-HCl pH 8.0, proteases and phosphatases inhibitors). For the immunoprecipitation step, 1 mg of pre-cleared protein extracts were incubated with 10 g of antibodies, along with 1 g of Spike-in antibody (Active Motif, #61686) and 50 ng of Spike-in chromatin from Drosophila (Active Motif, #53083) overnight at 4 °C with gentle rotation. For the negative control, Normal Rabbit IgG (Cell Signaling, #2729S) was employed. The next day, 50 l of Protein A/G PLUS-Agarose beads (Santa Cruz, #sc-2003) were added to each IP reaction and incubated for 2 h at 4 °C with rotation. The beads were washed twice with IP Dilution buffer supplemented with 0.1% SDS (final), 3 times with ChIP wash buffer (IP Dilution buffer plus 0.5 M NaCl, 0.1% SDS, and 1% [w/v] Sodium Deoxycholate), and once with TE buffer (10mM Tris-HCl, 1mM EDTA) containing 50 mM NaCl. Elution of immunocomplexes from beads was performed with Elution Buffer (50 mM Tris-HCl, pH 8; 10 mM EDTA, 1% SDS) and incubation at 65 °C for 15 min. Then, the supernatants were reverse crosslinked at 65 °C for 3 h, treated sequentially with RNAse A (Sigma-Aldrich, #R5503) at 37 °C for 2 h, and then treated with Proteinase K (New England Biolabs, #P8107S) at 55 °C for 2 h. DNA purification was performed with the QIAquick PCR Purification Kit (Qiagen, #28106). Purified DNA was used to generate libraries using the NEBNext Ultra DNA Library Prep Kit for Illumina (New England Biolabs, #7370) following the manufacturer's instructions.