GSM5171500: 0hour R1; Mus musculus; ChIP-Seq
Cells were cross-linked in medium containing 1% formaldehyde for 10 min at room temperature, neutralized with 0.125M glycine, scraped off and rinsed twice with cold 10 ml 1X PBS. Cells were pelleted by centrifugation for 7 min at 4°C at 600g. Pellets were resuspended in 10 mL of Buffer L1 (50mM Hepes KOH, pH 7.5, 140mM NaCl, 1mM EDTA pH 8.0, 10% glycerol, 5% NP-40, 0.25% Triton-X 100) and incubated at 4°C for 10 min. This was followed by centrifugation for 5 min at 4°C at 1300g. The pellet was again resuspended in 10 ml of Buffer L2 (200mM NaCl, 1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris pH 8.0) and incubated at room temperature for 10 min, followed by centrifugation for 5 min at 4°C at 1300g. The pellet was then resuspended in 900 µl Buffer L3 (1mM EDTA pH 8.0, 0.5mM EGTA pH 8.0, 10mM Tris pH 8.0, 100mM NaCl, 0.1% Na-deoxycholate, 0.17mM N-Lauroyl sarcosine) containing protease inhibitors and incubated at 4°C for overnight following sonication using Bioruptor plus (Diagenode). After clearing cellular debris by spinning at 14,000 g for 10 min at 4°C DNA 60 µg of chromatin were incubated at 4 °C overnight with respective antibodies after 1h of preclearing. The mixture was then incubated with 40 µl protein A-Sepharose beads preblocked with tRNA and BSA for 3 h at 4°C. Beads were washed twice with 1 ml buffer L3 and once with 1 ml DOC buffer (10 mM Tris (pH 8.0), 0.25 M LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA), and bound chromatin was eluted in 1% SDS/0.1 M NaHCO3. This followed treatment with RNase A (0.2mg/ml) for 30 min at 37°C and then with Proteinase K (50 µg/ml) for 2.5 h at 55°C. The crosslinking was reversed at 65°C overnight with gentle shaking. DNA was purified by phenol-chloroform extraction followed by ethanol precipitation and recovered in 40 µl TE buffer. ChIP-seq libraries were prepared for sequencing using standard Illumina protocols.
GEO Accession
GSM5171500