GSM2417329: Supt16_ChIP_seq_rep1,rep2; Mus musculus; ChIP-Seq
Cells were crosslinked with 1% formaldehyde for 20 min followed by quenching for 5 min with the addition of glycine to a final concentration of 0.125 M. After washing with PBS buffer, cells were collected and lysed in Cell Lysis buffer (5 mM Tris pH8.0, 85 mM KCl, 0.5% NP40 ) with proteinase inhibitors (10 µl/mL Phenylmethylsulfonyl fluoride (PMSF), 1 µl/mL Leupeptin and 1 µl/mL Pepstatin). Pellets were spun for 5 min at 5000 rpm at 4°C. Nuclei were lysed in Nuclei Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris HCl) and samples were sonicated for 12 min. 20 µL of 5 M NaCl were added and samples were reverse-crosslinked at 65°C for 4h. Samples were centrifuged for 20 min at 13,000 rpm at 4°C and the supernatant was diluted in IP buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris HCl, 167 mM NaCl) and the appropriate antibody was added and left overnight with rotation at 4°C. Protein A/G Dynabeads (Invitrogen) were added for 1h and after extensive washed samples were eluted in Elution Buffer (1% SDS, 0.1 M NaHCO3). Following phenol-chloroform extraction and ethanol precipitation, DNA was incubated at 37°C for 4h with RNAse (Sigma). Approximately 10-20 ng of ChIP material was used for library preparation. End-repair and adaptor ligation was prepared as described previously with a few modifications (Tessarz, et al). Double sided size selections (~200 – 650bp) were performed using the MagSI-NGS Dynabeads (MagnaMedics, #MD61021) according to the manufacturer’s instructions. Purified adapter-ligated ChIP material was run on a high sensitivity DNA chip on a 2200 TapeStation (Agilent) to assess size distribution and adaptor contamination.
GEO Accession
GSM2417329