title: GSM3271330: shRE JUND ChIP; Homo sapiens; ChIP-Seq
library_construction_protocol: Chromatin Immunoprecipitation was carried out as described previously. Cells were re-suspended at 3.3x106 cells/ml and crosslinked for 45 minutes with 0.83 mg/ml Di(N-succinimidyl) glutarate (DSG – sigma). Cells were washed four times with PBS before re-suspending at 2x106 cells/ml and crosslinking for 10 minutes with 1% formaldehyde (Pierce) in PBS. Reactions were quenched with 0.4 M glycine, washed twice with PBS and incubated with Buffer A (10 mM HEPES pH 8.0, 10 mM EDTA, 0.5 mM EGTA, 0.25% Triton X-100) at 4oC for 10 minutes followed by Buffer B (10 mM HEPES pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.01% Triton X-100, 0.25% SDS) at 4oC for 10 minutes. Chromatin was sheared (?cells/ml) in IP Buffer I (25 mM Tris 1 M, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1% TritonX-100 and 0.25 % SDS) using a PicorupterTM (Diagenode) to fragments of ~500bp. The supernatant was collected and diluted 3-fold in IP Buffer II (25 mM Tris, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1% TritonX-100, 7.5 % glycerol). Chromatin from 2x106 cells was incubated with 2 ug JunD coupled to 15 microliter protein G Dynabeads (Dynal) for 2 hours at 4oC. Beads were washed with Buffer 1 (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX-100, 0.1 % SDS), twice with Buffer 2 (20 mM Tris-HCl pH 8.0, 500 mM NaCl, 2 mM EDTA, pH 8.0, 1 % TritonX100, 0.1 % SDS), LiCl buffer (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 1 mM EDTA, pH 8.0, 0.5 % NP40, 0.5 % Na-deoxycholate) and twice with TE/NaCl buffer (10 mM Tris-HCl pH 8.0, 50 mM NaCl, 1 mM EDTA). Samples were eluted in 100 microliter elution buffer (100 mM NaHCO3, 1% SDS) and crosslinks were reversed overnight at 65oC with proteinase K. DNA was purified using Agencourt AMPure (Beckman Coulter) according to the manufacturer's instructions. Samples were analysed and validated by qPCR. DNA libraries from the chromatin immunoprecipitation assays were prepared using the Kapa Hyper-Prep kit according the manufacturer's guidelines. Libraries were indexed and then amplified by PCR. Libraries were gel purified to ensure the correct fragment lengths were sequenced (Qiagen Gel purification Kit). Fragments of 200-300bp were selected for DNase I libraries and 200-400bp for ChIP libraries. Libraries were validated by qPCR and quantified using the Kapa quantification kit before pooling and sequencing on NextSeq® 500/550 High Output kit v2 75 cycles (Illumina, FC 404-2005).
attributes.tag: GEO Accession
attributes.value: GSM3271330