GSM8787003: dTAG-SPT5-HCT116, ChIP_pSer2, muR2_Rep1; Homo sapiens; ChIP-Seq
About 20 million HCT116 cells grown on the 150 mm dish were cross-linked with 1% formaldehyde for 10 min at room temperature. Cross-linking was quenched with 125 mM glycine for 5 min. Cells were collected with a cell lifter (Costar) in 2 ml ChIP nuclei fractionation buffer [50 mM HEPES (pH 7.5), 140 mM NaCl, 1 mM EDTA (pH 8.0), 10% Glycerol, 1% NP-40, 0.25% Triton X-100] with proteinase and phosphatase inhibitors and incubated on ice for 10 min. Cell nuclei were collected by centrifugation at 4 °C at 600 × g for 10 min and washed with wash buffer [10 mM Tris-HCl (pH 8.0), 200 mM NaCl, 1 mM EDTA (pH 8.0), 0.5 mM EGTA (pH 8.0)]. The pellets were lysed in 1.8 mL RIPA buffer with proteinase and phosphatase inhibitors and sheared by sonication in a Bioruptor (Diagenode) at high power, for 2× 20 min with cycles of 30 s ON and 30 s OFF. Lysates were clarified by centrifugation at 16,000 × g for 10 min at 4 °C. 600 μL pre-cleared lysates were incubated with 4 μg antibodies overnight at 4 °C with end-to-end rotation. 22 μL Protein G magnetic beads (New England Biolabs) were added to the tube on the second day and the samples were incubated for another 4 hours at 4 °C with end-to-end rotation. The beads were washed with RIPA buffer three times, IP wash buffer [100 mM Tris-HCl (pH 8.0), 500 mM lithium chloride, 1% NP-40, 1% sodium deoxycholate] three times and TE buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)] twice. Reversal of cross-linking was done by incubating beads in the elution buffer [10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), 1% SDS, 200mM NaCl, 0.8 U Proteinase K] at 55 °C with heated lid for 16 h. On the third day, 1 μg of RNase A was added and the samples were incubated at 37 °C for 30 min. DNA was purified by using DNA Cleanup Kit (5 μg) (New England Biolabs). The purified DNA was used for qPCR and library preparation. Spike-In DNA (S. cerevisiae) (Cell Signaling Technology) were added to each sample and DNA libraries were prepared with NEBnext Ultra II DNA library kit (New England Biolabs).