GSM1979824: HuRef_CenpA_(20141219_1); Homo sapiens; ChIP-Seq
The HuRef lymphoblastoid cell  line was grown in RPMI media  using standard protocols. Antibodies used in this study were purchased from Abcam (Cambridge MA). Nuclei were prepared following a previously published protocol (PMID: 23644596). Briefly, for each IP 40-60 million cells were collected and washed with 1X PBS. Cells were re-suspended in ice-cold buffer I (0.32 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF and protease inhibitor) at a density equal to ~25 million cells/ml. An equal volume of ice-cold buffer I supplemented with 0.1% NP40 was added and samples were incubated on ice for 10 min. Nuclei solution was layered on ice-cold buffer III (1.2 M sucrose, 60 mM KCl, 15 mM NaCl, 5 mM MgCl2, 0.1 mM EGTA, 15 mM Tris, pH 7.5, 0.5 mM DTT, 0.1 mM PMSF and protease inhibitor) and centrifuged at 10,000g for 20 min at 4 °C. The pellet was re-suspended in buffer A (0.34 M sucrose, 15 mM HEPES, pH 7.4, 15 mM NaCl, 60 mM KCl, 4 mM MgCl2, 1 mM DTT, 0.1 mM PMSF and protease inhibitor, 3mM Cacl2) at density equal to ~ 32.5 million cells/ml.Micrococcal nuclease (MNase, Sigma, Cat # N3755) was added to ~2.5 units/ml, and digestion was carried out at 37°C for 5 min. Reactions were stopped by addition of EGTA to a final concentration of 20 mM and EDTA to 10 mM. The final NaCl concentration was adjusted to 215 mM and needle extraction was performed as described previously (PMID: 22184235) to enhance the solubility of kinetochore complex. The resulting solution was incubated overnight at 4 °C on nutator. Soluble chromatin was collected by centrifuging the mixture at 12000rpm at 4 °C for 8 min. Soluble chromatin was diluted 3X with 20 mM Tris, pH 8.0, 5 mM EDTA and 200 mM NaCl, and Triton-X was added at a final concentration of 0.1% (v/v). Next, the chromatin solution was pre-cleared using Protein A/G fast flow Sepharose beads for 20 min at 4 °C, and 15 µg of antibody (Abcam anti-CENP-A Ab13939) was added per ChIP sample and incubated overnight at 4 °C. Dynabeads were added to the samples and incubated at 4°C for 2 hrs. Immunoprecipitated complexes were washed 6 times with 50mM Phosphate buffer pH 7.4, 5mM EDTA, 200mM NaCl, and DNA was extracted from Dynabeads.
GEO Accession
GSM1979824