GSM5593294: Ch12 WT biotag Smad7 rep3; Mus musculus; ChIP-Seq
ChIP-Seq: Cultured cells were fixed with 1% formaldehyde (Sigma) for 10' at 37°C. Fixation was quenched by addition of glycine (Sigma) at a final concentration of 125 mM. Twenty million fixed cells were washed with PBS and stored at −80°C until further processing or resuspended in 800 uL of RIPA buffer (10 mM Tris pH 7.6, 1 mM EDTA, 0.1% SDS, 0.1% sodium deoxycholate, 1% Triton X-100, 1 × Complete Mini EDTA free proteinase inhibitor (Roche)). Sonication was performed using Bioruptor sonicator, 30 cycles of 30” sonication and 30” of pause. Five microgram of respective antibody was incubated with 40 μL of Dynabeads Protein A (or G) for 40 min at room temperature. Antibody-bound beads were added to 500 μL of sonicated chromatin, incubated at 4°C overnight, and washed twice with RIPA buffer, twice with RIPA buffer containing 0.3M NaCl, twice with LiCl buffer (0.25 M LiCl, 0.5% Igepal-630, 0.5% sodium deoxycholate), once with TE (pH 8.0) plus 0.2% Triton X-100, and once with TE (pH 8.0). Crosslinking was reversed by incubating the beads at 65°C for 4 hr in the presence of 0.3% SDS and 1 mg/mL Proteinase K. ChIP DNA was purified by ChIP DNA clean and concentrator column (Zymo research). ChIP-Seq: Library was prepared in Ovation SP Ultralow library system (Nugen). 50 cycles of sequencing data were acquired on Illumina HiSeq 3000.