GSM9039686: ChIP-SMAD2/3-ctrl; Homo sapiens; ChIP-Seq
ChIP assay was performed using SimpleChIP Enzymatic chromatin IP Kit (CST, 9003S). HUVECs were fixed with 1% formaldehyde for 10 min, followed by quenching in 125 mM glycine. After washing two times in ice-cold PBS, the cell pellest were lysised in 8 ml buffer A on ice. The cell nuclei were resuspended in 8 ml buffer B, then washed two times in 1 ml ChIP buffer and sonicated for 10 min using covaris S220. 10 μg chromatin were suspended in 500 ul ChIP buffer with proteinase inhibitor, supplied with pretreated protein G magnetic beads at 4°C for 1 h. The chromatin supernatant were added with anti-SMAD2/3 (Abcam, ab202445) and incubated at 4°C overnight. The bound chromatin were further combined with pretreated protein G magnetic beads at 4°C for 2 h, then washed three times with ChIP buffer and one time with ChIP buffer supplemented with 0.3 M NaCl. The complex of chromatin and magnetic beads were resuspended in 150 ul ChIP elution buffer and eluted by 1200 rpm at 65°C for 30 min. IP product were finally de-crosslinked by RNase A at 37°C for 2 h and proteinase K at 65°C overnight. ChIP-seq libraries were prepared according to the standard protocol of NEBNext Ultra DNA library Prep Kit (NEB, E7645B).