GSM5287884: SHP-1-KO_WCE (input DNA); Homo sapiens; ChIP-Seq
Cells were washed 3 times with PBS and 5x107 cells for each ChIP-seq fixed with 1% formaldehyde for 10 min. Cross-linked cells were quenched using glycine for 5 min at room temperature. Cells were washed twice with ice-cold PBS and harvested using a silicon scraper. Cells were transferred to 50 ml falcon and centrifuged at 1350 g for 5 min. Pellets obtained were washed again with ice-cold PBS. Cell pellets were suspended in LB1 buffer (50 mM Hepes-KOH, pH7.5, 140 mM NaCl, 1 mM EDTA, 10 % glycerol, 0.5% IGEPAL, 0.25% Triton X-100, PMSF and protease inhibitor cocktail) and incubated at 4°C for 10 min with rotation. Pellets were obtained using centrifugation. The cell pellet was suspended in LB2 buffer (10 mM Tris-HCl, pH8.0, 200 mM NaCl, 1mM EDTA, and 0.5 mM EGTA supplemented with protease inhibitors) and incubated for 10 min at room temperature with rotation. Lysates were centrifuged at 1350 g for 5 min and pellets were suspended in sonication buffer (20 mM Tris-HCl pH 8.0, 150 mM NaCl, 2 mM EDTA pH8.0, 0.1 % SDS, and 1% Triton X-100 supplemented with proteinase inhibitors). Lysates were sonicated using Bioruptor instrument (Diagenode) for a total of 6 min (high intensity, 12 cycles of 30 sec ON and 30 sec OFF) at 4°C to obtain a chromatin fragmentation of 200-900 bp. Protein G dynabeads pre-blocked in blocking solution (0.5% BSA in PBS) were incubated with antibodies (RPB1 and pSer2-RPB1) at 4°C for 6 h. Protein G Dynabead-antibody complexes were washed 3 times with 1 ml blocking solution, then added to chromatin preparations and incubated overnight at 4°C. Some portions of the chromatin were left untreated and used as whole-cell extract (WCE) control.  Beads were washed once with Wash buffer B (20 mM Tris-HCl pH8.0, 150 mM NaCl, 2 mM EDTA pH8.0, 0.1% SDS, 1% Triton X-100 and protease inhibitors), followed by wash buffer C (20 mM Tris-HCl pH8.0, 500 mM NaCl, 2 mM EDTA pH8.0, 0.1% SDS, 1% Triton X-100 and protease inhibitors), and Wash Buffer D (10 mM Tris-HCl pH8.0, 250 mM LiCl, 1 mM EDTA pH8.0, 1% NP40 and protease inhibitor) and TE buffer (10 mM Tris pH8.0, 1 mM EDTA, 50 mM NaCl and protease inhibitors). Complexes were eluted off the beads using elution buffer containing 50 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0, and 50 mM NaCl. Beads were collected and incubated at 65°C overnight to reverse cross-linked. In parallel, WCE were also reverse cross-linked.  Samples were subsequently treated with RNase A (Ambion) at 37°C for 2 hours followed by Proteinase K (InVitrogen) treatment at 55°C for 30 min. DNA was purified using the phenol-chloroform method. Libraries were constructed from ChIP- and WCE-samples using NEBNext Ultra II DNA library prep kit (New England Biolabs) according to the manufacturer's instructions and single-end sequenced (50 bp) on an Illumina HiSeq 2500 instrument at the Next-Generation Sequencing Platform of the CHU de Québec-Université Laval Research Center (CRCHUQ).