GSM6532766: ChIP_seq_FoxO1_HA_DMSO; Homo sapiens; ChIP-Seq
Cells were washed once with cold PBS prior to cross-linking with 2 mM DSG for 45 minutes followed by 1% formaldehyde (28908, Thermo) for 15 min at room temperature (except for the etoposide ChIP of TOP2A, cells were treated with 100mM etoposide for 10min). To quench the cross-linking reaction, 2.5 M glycine dissolved in PBS was added and cells were incubated at room temperature for a further 5 minutes. Cells were washed twice with ice-cold PBS and lysed using cell lysis buffer (50 mM Tris pH 8.0, 0.5% NP40, 50 mM NaCl) to remove the cytoplasmic proteins. The pellet was resuspended in nuclear lysis buffer (1% Triton X-100, 2 mM EDTA pH 8.0, 400 mM NaCl, 20 mM Tris pH 8.0, 0.1% SDS). Chromatin DNA was sheared to 300–500 bp average in size through sonication using a Bioruptor instrument (Diagenode). Fragmented chromatin was diluted 1:1 with dilution buffer (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 1% Triton X-100) and immunoprecipitated with IgG or indicated specific antibodies overnight at 4°C, followed by incubation with protein A/G magnetic Dynabeads for 4 h. Dynabeads were washed once each with ChIP wash buffer 1 (20 mM Tris pH 8.0, 150 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100) and ChIP wash buffer 2 (20 mM Tris pH 8.0, 400 mM NaCl, 2 mM EDTA pH 8.0, 0.1% SDS, 1% Triton X-100) and washed twice with TE buffer (10 mM Tris pH 7.5, 1 mM EDTA pH 8.0). After washing and elution, the protein–DNA complex was reversed by heating overnight at 65°C. Immunoprecipitated DNA was purified by QIAquick spin columns. Sequencing libraries were prepared using the KAPA DNA HyperPrep Kit and subjected to high-throughput sequencing. Sequencing libraries were prepared using the KAPA DNA HyperPrep Kit and subjected to high-throughput sequencing. Briefly, Chip DNA was end-blunted and added with an 'A' base. Then 200–400 bp fragments are gel-isolated and purified. The library was amplified by 18 cycles of PCR.