GSM7506239: TS186_05_shx_H3K27ac_THP1_S5; Homo sapiens; ChIP-Seq
100x10^6 cells were treated for 1-, 2-, 6- and/or 48- hours with CCS1477. Cells were counted and cross-linked using 1% formaldehyde (F8775, Sigma Aldrich) in PBS for 10 minutes (for H3K27ac), or double cross-linked (for EP300, MYB, IRF4, CBP, TCF3/E2A and RUNX1) with ChIP Cross-link Gold (C01019027, Diagenode) for 30 minutes in PBS with 1mM MgCl2 and then with 1% formaldehyde for 10 minutes. The reaction was stopped by addition of 0.125M glycine for 5 minutes. Cell pellets were then washed twice with cold PBS containing protease inhibitors (Complete EDTA-free tablets, 11873580001, Roche). ChIP was performed as described in Lee et al. (2006, doi: 10.1038/nprot.2006.98). Briefly, nuclear lysates were sonicated by using a Bioruptor Pico for 8 and 16 cycles for THP1 and OPM2, respectively (30 seconds ON, 30 seconds OFF settings). Chromatin Immunoprecipitation was carried out overnight at 20 rpm, 4°C in a rotating wheel with 180μl Dynabeads Protein G (10004D, Invitrogen) previously conjugated with 15μg antibody. Beads were then washed six times with the RIPA buffer (50mM HEPES pH7.5, 1mM EDTA, 0.7% Na deoxycholate, 1% NP-40, 0.5M LiCl) and once with TE buffer (10mM Tris pH8.0, 1mM EDTA). Later, chromatin IP-bound fractions were extracted and de-crosslinked with Odom elution buffer (50mM TrisHCl pH8.0, 10mM EDTA, 1% SDS) and incubated in a thermomixer at 65°C, 900 rpm overnight.  The day after, RNaseA (0.2mg/ml) and proteinase K (0.2 μg/ml) were added to remove any RNA and protein from the samples. Finally, DNA was extracted using phenol:chloroform:isoamyl alcohol extraction and precipitated with ethanol: two volumes of ice-cold 100% ethanol, glycogen (20 μg/μl) and 200mM NaCl were added to each sample, which were then incubated overnight at −80°C. Pellets were washed with 70% ethanol and resuspended in 30μl 10mM TrisHCl pH8.0. Libraries were prepared for sequencing using NEBNext UltraII (New England Biolabs) starting from 1-5ng of ChIP DNA, according to the manufacturer's instructions. Fragments of 200–800 bp were selected using AMPure beads (Beckman Coulter, Pasadena, CA). Sequencing was performed using either a NovaseqSP (2x100 cycles) or a Novaseq 2x150 with 100-bp, paired-end high output generating between 50-90 million reads per sample.