GSM2702905: CTCF ChIPSeq TPO 30 minutes Rep 1; Mus musculus; ChIP-Seq
Chromatin immunoprecipitation (ChIP) assays on serum-starved and TPO stimulated HPC7 cells were performed as previously described (Wilson et al. 2010). Briefly, 10^8 cells were cross-linked in 1% formaldehyde for 10 min at room temperature. Cells were lysed in 10mM Tris pH 8.0, 10mM NaCl and 0.2% NP40 containing inhibitors (1g/mL leupeptin, 10mM NaBu and 50g/mL PMSF) for 10 min on ice and were collected by centrifugation at 2500 rpm for 5 min at 4ºC. The nuclei pellet was frozen until further use. Frozen nuclei were resuspended in 50mM Tris pH 8.0, 10mM EDTA, 1% SDS supplemented with inhibitors (1g/mL leupeptin, 10mM NaBu and 50g/mL PMSF) and sonicated in an equal volume of IP dilution buffer (20mM Tris pH 8.0, 2mM EDTA, 150mM NaCl, 1% Triton X-100, 0.01% SDS and protease inhibitors) in ice-water (Bioruptor, Diagenode) for 5 cycles (30s on, 30s off). Chromatin was pre-cleared with non-specific rabbit IgG (2 μg/μl, Sigma) for 1 hour and 100μL of protein G Dynabeads (Invitrogen) for 2 hours. The beads/IgG were removed by magnetic separation. Chromatin was immunoprecipitated at 4ºC overnight using antibodies against H3K27ac (Abcam, 4729), Rad21 (Abcam, 992), CTCF (Millipore, 07-729), STAT1 (Cell Signaling, 9172) or a control rabbit IgG (Invitrogen, 9172) and 100μl of protein G Dynabeads (Invitrogen) were added for additional 2 hours. Immunocomplexes were washed and eluted twice from the beads with 150μL elution buffer (100mM NaHCO3, 1% SDS). Cross-linking was reversed overnight with 0.3M NaCl and 2μL of RNase (10mg/ml) at 65ºC, and samples were further treated with Proteinase K (20mg/ml) for 2 hours at 42ºC. The ChIP DNA was purified using a PCR purification kit (Qiagen). Libraries were prepared according to Illumina's instructions accompanying the DNA and RNA Sample Kit. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on a HiSeq4000 sequencer.