GSM1979830: HT10801b_CenpA_CenpT_X1_(20151015_5); Homo sapiens; ChIP-Seq
The HT1080-1b fibrosarcoma cell  line was grown in DMEM media  using standard protocols. Antibodies used in this study were purchased from Abcam (Cambridge MA). Primary cross-linking ChIP was performed as described previously (PMID: 24737864) with few modifications. Briefly, for each IP 200 million cells were fixed at a density equal to 2 million/ml using formaldehyde to a final concentration of 1%. Cross-linking was quenched with 125mM glycine. Chromatin was released from cells by incubating in lysis buffer (1% SDS, 10mM EDTA and 50 mM Tris-HCl (pH 8.1)) on ice for 10 min. The resulting chromatin solution was diluted 10 times with IP dilution buffer (1% Triton X-100, 2mM EDTA, 150 mM NaCl and 20mM Tris-HCl (pH8.1)) and digested with Micrococcal nuclease (MNase, Sigma, Cat # N3755) at 37°C for 45 min. MNase digested chromatin was sonicated as described before ( PMID: 24737864)and  solubilized chromatin was precleared using protein A sepharose beads for 30 minutes at 4 °C. The precleared chromatin was incubated with Anti-FLAG-M2 magnetic beads (Sigma Cat # M8823) for 4 hrs at 4 °C . The FLAG-M2 magnetic beads were washed  once with buffer I (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1), and 150 mM NaCl), four times with buffer II 0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl (pH 8.1) and 150 mM NaCl), 2 times with buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA and 10 mM Tris-HCl (pH 8.1)) and once with 1X TE. Protein complexes were eluted from magnetic beads using FLAG peptide in HE buffer (10 mM HEPES, 0.25 mM EDTA, 0.5 mM PMSF). The eluent was subjected to subsequent ChIP reaction with Anti-CENP-T (Abcam, Cat# Ab114120)antibodies.Solexa library construction was performed as described previously (PMID:22025700) and PCR-amplified using Phusion polymerase.
GEO Accession
GSM1979830