GSM5708667: CIC-KO1 (IDH1-R132H) H3K4me3 rep 1; Homo sapiens; ChIP-Seq
Samples for Native chromatin immunoprecipitation (N-ChIP) were prepared from ~100,000 cells per immunoprecipitation, two replicates per each cell line. In brief, cells were lysed using lysis buffer (0.1% Triton X-100, 0.1% deoxycholate) and protease inhibitor for 20 min on ice. The extracted chromatin was then digested using 90 U of MNase enzyme (NEB) for 6 min at 25 °C. Reactions were quenched by adding 5.5 µL of 250 µM EDTA. A mix of 1% Triton X- 100 and 1% deoxycholate was then added to the digested samples and the 96-well plate of samples was chilled on ice for 20 min. The digested chromatin was then pooled and 12 µl of chromatin was reserved for use as input control. The rest of the digested chromatin was pre- cleared using IP buffer (20 mM Tris-HCl [pH7.5], 2 mM EDTA, 150 mM NaCl, 0.1% Triton X-100, 0.1% deoxycholate) plus protease inhibitor with 20 µl of pre-washed Protein A/G Dynabeads (Invitrogen) at 4 °C for 1.5 h. Supernatants were removed from the beads and transferred to a 96-well plate containing the antibody-bead complex. The plate was sealed and incubated at 4 °C on a rotating platform overnight. The reaction plate containing the immunoprecipitation samples was placed on a magnetic plate and samples were washed twice with low salt buffer (20 mM Tris-HCl [pH 8.0], 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 150 mM NaCl) and twice with high salt buffer (20 mM Tris-HCl [pH 8.0], 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 500 mM NaCl). DNA-antibody complexes were eluted in 30 µl Elution Buffer (100 mM NaHCO3, 1% SDS), incubated at 65 °C for 1.5 h with mixing at 1,350 rpm on a thermomixer. Protein was digested by adding 1.75µl of Qiagen Protease to the eluted DNA samples at 50 °C for 30 min with mixing at 600 rpm on a thermomixer. ChIP DNA was then purified using Sera-Mag beads (ThermoFisher Scientific) with 30% PEG before library construction. Library construction and sequencing were done identically to the CIC ChIP-seq samples with the only difference being that the adaptor-ligated products were amplified using 8 to 10 PCR cycles.