GSM6621079: BATF-IP_1; Homo sapiens; ChIP-Seq
Ten million cells were washed twice in cold PBS buffer and cross-linked with 1% formaldehyde for 10 minutes at room temperature and then quenched by addition of glycine (125 mmol/L final concentration). Afterwards, samples were lysed and chromatins were obtained on ice. Chromatins were sonicated to get soluble sheared chromatin (average DNA length of 200-500 bp). 20 µL chromatin was saved at -20°C. For input DNA, 100 µL chromatin was used for immunoprecipitation by anti-BATF antibodies (Cat# 8638S，Cell Signaling Technology) and IgG antibodies (ab17870, Abcam) respectively. 10 μg of antibody was used in the immunoprecipitation reactions at 4°C overnight. The next day, 30 μL of protein beads was added and the samples were further incubated for 3 h. The beads were next washed once with 20 mM Tris/HCL (pH 8.1), 50 mM NaCl, 2 mM EDTA, 1% Triton X-100, 0.1% SDS; twice with 10 mM Tris/HCL (pH 8.1), 250 mM LiCl, 1 mM EDTA, 1% NP-40, 1% deoxycholic acid; and twice with TE buffer 1× (10 mM Tris-Cl at pH 7.5. 1 mM EDTA). Bound material was then eluted from the beads in 300 μL of elution buffer (100 mM NaHCO3, 1% SDS), treated first with RNase A (final concentration 8 μg/mL) during 6 h at 65°C and then with proteinase K (final concentration 345 μg/mL) overnight at 45°C. Immunoprecipitated DNA was used to construct sequencing libraries following the protocol provided by the I NEXTFLEX® ChIP-Seq Library Prep Kit for Illumina® Sequencing (NOVA-5143-02，Bioo Scientific) and sequenced on Illumina Xten with PE 150 method.