GSM3288351: 17_MYB_shRVBL2; Homo sapiens; ChIP-Seq
25x106 shSCR or shRUVBL2 transduced THP1 cells 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 15 minutes at room temperature and the reaction quenched by adding glycine to final concentration 0.25 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 on ice each time. Cells were finally resuspended in incubation buffer (0.15% SDS, 1% TritonX100, 150 nM NaCl, 10 mM EDTA, 0.5 mM EGTA, 20 mM HEPES [pH 7.6] ) plus protease inhibitors (Sigma Aldrich) and incubated for 30 minutes on ice. Lysates were then sonicated for 6 cycles (30 sec on / 30 sec off) in the Bioruptor® Pico water bath sonicator (Diagenode, Liege, BE). Cleared supernatant was stored at -80°C. 500 μl of sonicated chromatin were diluted 10 times in ChIP Dilution Buffer (0.01% SDS, 1% Triton X-100, 1.2 mM EDTA [pH 8], 16.7 mM Tris-HCl pH 8 and 167 mM NaCl) and were incubated rotating at 4°C overnight, with protein A/G magnetic beads (Merck Millipore) and 2-10 μg of anti-MYB (1-1, Merck Millipore; and D-7, Santa Cruz Biotechnology) or anti-H3K27Ac (ab4729, Abcam) antibodies. Beads were washed for 10 minutes once in low salt buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA pH 8, 20 mM Tris-HCl [pH 8.1], 150 mM NaCl), in high salt buffer (0.1% SDS, 1% TritonX100, 2 mM EDTA, 20 mM Tris-HCl [pH 8.1], 500 mM NaCl), LiCl Buffer (0.25 M LiCl, 1% NP-40, 1 mM EDTA, 10 mM Tris-HCl pH 8.1) and in TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA). DNA was eluted from the ChIP samples by two 1 hour incubations at 55°C 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 prepped 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. Libraries were prepped 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.