GSM6594091: LG02_REH_DMSO_ChIP_TP53; Homo sapiens; ChIP-Seq
REH cells were treated for 6h with either S3I-201 (50 uM) or DMSO and then were cross-linked with 2 mM DSG for 30 minutes at room temperature. After washing the samples 3 times with PBS, cells were fixed with 1% formaldehyde for 10 minutes at room temperature. The reaction was quenched by adding glycine to final concentration 0.125 M for 5 minutes at room temperature. Fixed cells were washed twice with cold PBS, and then sequentially in lysis buffer A (0.25% TritonX100, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES [pH 7.6]) and lysis buffer B (150 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES [pH 7.6], incubating for 10 minutes in  rotation at 4°C each time. Cells were then resuspended in incubation buffer (0.15% SDS, 1% TritonX100, 150 mM NaCl, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES [pH 7.6]) plus protease inhibitors (Roche, Merck) and incubated for 30 minutes on ice. Lysates were then sonicated for 20 cycles (30 sec on / 30 sec off) in the Bioruptor Pico water bath sonicator (Diagenode, Liege, BE). Cleared supernatant was stored at -80°C. 250 μl of sonicated chromatin were diluted in incubation buffer supplemented with 0.1% BSA and protease inhibitors and were incubated rotating at 4°C overnight, with protein A magnetic beads (Merck Life Science) and 2 ug of anti-TP53 (C15410083  ,Diagenode) antibody. Beads were washed for 10 minutes twice in low salt buffer (0.1% SDS, 0.1% DOC, 1% Triton X-100, 0.1 mM EDTA pH 8.0, 0.5 mM EGTA, 10 mM Tris-HCl [pH 8.0], 150 mM NaCl), and once in high salt buffer (0.1% SDS, 0.1% DOC, 1% Triton X-100, 0.1 mM EDTA pH 8.0, 0.5 mM EGTA, 10 mM Tris-HCl [pH 8.0], 500 mM NaCl), LiCl Buffer (250 mM LiCl, 0.5% DOC, 0.5% NP-40, 0.1 mM EDTA, 0.5 mM EGTA, 10 mM Tris-HCl [pH 8.0]) and in TE buffer (10 mM Tris-HCl [pH 8.0], 0.1 mM EDTA, 0.5 mM EGTA). DNA was eluted from the ChIP samples by 20 minutes incubation in rotation at RT with elution buffer (1% SDS, 100 mM NaHCO3). Chromatin was reverse-crosslinked and subjected to RNase and proteinase K digestion and extracted by using the MinElute PCR purification kit (Qiagen). Libraries were prepared from 1ng of ChIP DNA using the NEBNext DNA Ultra II assay (New England Biolabs, Hitchin, UK) with bead-based size selection for 200 bp fragments and 12 cycles of amplification. Samples were then sequenced on an Illumina NextSeq 500, using a 43bp, paired end configuration.