title: GSM9242319: input, DMSO; Homo sapiens; ChIP-Seq
library_construction_protocol: Approximately 20 x 106 cells were harvested per IP and fixed with 1% methanol-free formaldehyde for 8 min. Crosslinking was quenched by adding glycine to a final concentration of 0.125mM at 4°C for 10 minutes. Cells were washed twice with ice-cold PBS. After washing, cells were harvested by scraping in ice-cold PBS containing proteinase inhibitor cocktail (Roche) and pelleted at 600 × g for 7 min at 4°C. Cells were incubated for 10 min on ice in a buffer containing 10 mM EDTA, 10 mM Tris pH 8, 0.5 mM EGTA and 0.25% Triton X-100. After centrifugation, cells were incubated in 1 mM EDTA, 10 mM Tris pH 8, 0.5 mM EGTA, 200 mM NaCl and passed 3 times through 26-G needles and incubated on ice for 10 minutes. Cells were centrifuged for 600 × g for 7 min at 4°C. Chromatin was extracted in a high-salt buffer containing 50 mM HEPES, pH 7.5, 1 mM EDTA, 1% Triton X-100, 0.1% deoxycholate, 0.1% SDS and 500 mM NaCl for 2h at 4 °C. Chromatin was sheared using a Bioruptor Pico sonicator (Diagenode). Per IP 50 ul of protein-G Dynabeads (Invitrogen) were washed twice with TE buffer pH 8 for 5 minutes at room temperature. Beads were blocked with 1 mg BSA (Sigma), 100 ng yeast tRNA (Sigma) in TE buffer containing proteinase inhibitor cocktail. 100 µg of chromatin was pre-cleared with 20ul of pre-blocked protein-G magnetic beads for 1 hour at 4°C. Then, 5% of cleared-chromatin was stored at -20 °C as input until de-crosslinked together with the IP-material. For the IP, 1 μg of anti-γH2AX (Sigma) or 7,5 μg anti-GFP (Abcam) was added to cleared chromatin and incubated overnight at 4 °C. The following day, 30 µl blocked protein-G beads was added to the chromatin and incubated for 3h at 4°C. Beads were separated by magnet and washed twice for 8 min with high-salt buffer, once for 8 minutes with 50 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, and twice with TE buffer pH 8, on overhead rotator at 4 °C. Next, chromatin was eluted in 1% SDS and 100 mM NaHCO3 containing 60 µg RNaseA (Roche) and incubated at 37 °C for 30 min. Elution was adjusted to 10mM EDTA, 40mM Tris pH 8.0, and 25µg Proteinase K (Roche) and incubated for 3h at 55°C. Final decrosslinking was done overnight at 65°C. Input-chromatin was de-crosslinked in parallel in TE buffer pH 8 and 60 µg RNaseA was added and incubated at 37°C for 30 min. Chromatin was adjusted to 1% SDS, 100mM NaCl and 60µg Proteinase K and incubated for 3h at 55°C. Final decrosslinking was done overnight at 65°C. Eluted material was cleaned up by phenol-chloroform extraction and DNA was quantified using a Qubit 3.0 fluorometer (Thermo Scientific) ChIP-seq libraries using the NEBNext Ultra II DNA Library Prep kit for Illumina (E7645) following the standard protocols and using 10 ng of DNA. Samples with different index barcodes were combined at equal molar ratios, with the exception of input DNA (0.7x ratio), and sequenced as pools.
identifiers.namespace: GEO
identifiers.id: GSM9242319_r1