GSM4040259: CHD6m.a.mut_ChIPSeq_iPSC; Homo sapiens; ChIP-Seq
For each batch of ChIP experiments ~12 million cells were crosslinked in 2% PFA for 45 min at 4°C. From this point onward, cells were processed via the ChIP-IT High Sensitivity kit (Active motif) as per manufacturer's instructions, but using the NEXSON protocol for the isolation of nuclei [1]. Chromatin was sheared to 200-500 bp fragments on a Bioruptor Plus (Diagenode; 2x 20-26 cycles of 30s “on” and 30s “off”, at the highest power setting), and immunoprecipitations were carried out by adding 4 μg of the appropriate antiserum (CHD6, A301-221A Bethyl; TF3B, A303-673A Bethyl) to ~30 μg of chromatin and incubating on a rotator 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) and used in qPCR or sequencing on a HiSeq4000 platform (Illumina). For each batch of ChIP experiments ~12 million cells were crosslinked in 2% PFA for 45 min at 4°C. From this point onward, cells were processed via the ChIP-IT High Sensitivity kit (Active motif) as per manufacturer's instructions, but using the NEXSON protocol for the isolation of nuclei [1]. Chromatin was sheared to 200-500 bp fragments on a Bioruptor Plus (Diagenode; 2x 20-26 cycles of 30s “on” and 30s “off”, at the highest power setting), and immunoprecipitations were carried out by adding 4 μg of the appropriate antiserum (CHD6, A301-221A Bethyl; TF3B, A303-673A Bethyl) to ~30 μg of chromatin and incubating on a rotator 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) and used in qPCR or sequencing on a HiSeq4000 platform (Illumina).