title: GSM2936332: IMR90_HMGB1_ChIP (proliferating); Homo sapiens; ChIP-Seq
library_construction_protocol: For each batch of ChIP experiments approx. 25 million cells, cultured to >80% confluence in 15-cm dishes, were crosslinked in 15 mM EGS/PBS (ethylene glycol bis(succinimidyl succinate); Thermo Scientific) for 20 min at room temperature, followed by fixation for 40 min at 4°C in 1% PFA. From this point onwards, cells were processed using the ChIP-IT High Sensitivity kit (Active motif) as per manufacturer's instructions. Chromatin was sheared to 200-500 bp fragments on a Bioruptor Plus (Diagenode; 2x 9 cycles of 30 sec on and 30 sec off, at the highest power setting), and immuno-precipitation was carried out by adding 4 μg of the appropriate antiserum (HMGB1: 4F10, DSHB, HMGB2: ab67282, Abcam; CTCF: 61311, Active motif) to approx. 30 μg of chromatin and incubating on a rotaror overnight at 4°C in the presence of protease inhibitors. Following addition of protein A/G agarose beads and washing, DNA was purified using the ChIP DNA Clean & Concentrator kit (Zymo Research). DNA libraries were prepared using the Illumina TruSeq RNA sample preparation Kit v2 starting from the end repair step of the protocol. Up to 20 ng ChIP DNA was used as starting material. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified for 18 PCR cycles to create the final libraries.
attributes.tag: GEO Accession
attributes.value: GSM2936332