GSM7316270: sw480_smad4_bir_TAGCTT.june2015; Homo sapiens; ChIP-Seq
Each 100 μl cell pellet was incubated with 1 ml of cross-linking solution containing an equal amount of DSG (Thermo), DSS (Thermo) and EGS (Thermo 21565), creating a final concentration of 2 mM NHS ester (reactive groups). Cells were rocked for 20 min at room temperature, after which fresh formaldehyde was added to make a concentration of 1.22% formaldehyde. The cells were then rocked for an additional 20 min. The cross-linking reaction was stopped with 125 mM glycine. The cells were then washed with PBS, and nuclei were extracted using a Dounce homogenizer and Farnham lysis buffer (5 mM PIPES, pH 8.0, 85 mM KCl, 0.5% NP-40, and protease inhibitor cocktails). IP material was subsequently handled as described above for conventional ChIP of sonicated genomic DNA (a final concentration of 0.2% SDS in the sonicates). The diluted sonicates were then incubated with pre-blocked (0.5% BSA/PBS) Streptavidin beads (Invitrogen) overnight at 4°C. The beads were serially washed in low salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 150 mM NaCl, 1 mM EDTA, and 20 mM HEPES, pH 8.0), high-salt (0.1% SDS, 0.1% Sodium Deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 20 mM HEPES, pH 8.0), LiCl buffer (250 mM LiCl, 0.5% Sodium Deoxycholate, 0.5% NP-40, 1mM EDTA, and 20 mM HEPES, pH 8.0), and a final wash buffer (1 mM EDTA and 20 mM HEPES, pH 8.0). The beads were then incubated at 65 °C for 6 h in reverse cross linking buffer (0.1 mM NaHCO3 and 1% SDS). The DNA was purified by QIAquick PCR Purification Kit (QIAGEN) and quantified with PicoGreen (Life Technologies). 5 ng each of ChIP or input DNA was used to prepare ChIP-seq libraries with a Rubicon Genomics ThruPLEX DNA-seq Kit (Illumina).