GSM8172208: ChIP-Seq_GapR-HA(High)_hTERT-RPE1; Homo sapiens; ChIP-Seq
PBS, cells were resuspended and lysed for 10 min at 4°C in 1 mL Cell Lysis Buffer (20 mM Tris-HCl (pH 8.0), 85 mM KCl, 0.5% NP-40) supplemented with EDTA-free protease inhibitor cocktail (Roche, 4693132001). The nuclei were recovered by centrifugation at 500 g for 5 min at 4°C and washed once in the same buffer. Nuclei were resuspended in 500 μL Nuclei Lysis Buffer (10 mM Tris-HCl (pH 7.5), 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, protease inhibitor cocktail) and incubated for 10 min at 4°C. Chromatin was fragmented by sonication 3 × 10 s at 60% duty cycle, with 30 sec on wet-ice between sonication cycles, using a microtip-equipped SLPe sonifier (Branson Ultrasonics ™ Sonifier™, Brookfield, USA). The sonicated samples were centrifuged at 16,000 g for 10 min. The supernatants (500 µL) were collected and diluted with 1500 μl ChIP Dilution Buffer (0.01% SDS, 1.2 mM EDTA, 1.1% Triton X-100, 16.7 mM Tris-HCl (pH 8), 167 mM NaCl) supplemented with protease inhibitors and mixed with 50 μL Dynabeads™ Protein G (Invitrogen, 10009D) coupled with 3 μg anti-HA antibody (Invitrogen, MA5-27915). After overnight incubation at 4°C on a rotating wheel, the beads were washed twice with 1 mL Cell Lysis Buffer, twice with high-salt buffer (50 mM HEPES-KOH (pH 7.5), 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), once with LiCl buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA, 250 mM LiCl, 1% NP-40, and 1% sodium deoxycholate) and once with TE buffer (10 mM Tris-HCl (pH 8.0), 1 mM EDTA). To elute immunoprecipitated chromatin fragments, beads were incubated for 10 min at 65°C in 400 μL freshly prepared elution buffer (1% SDS, 0.1 M NaHCO3). DNA crosslinks were reversed by overnight incubation at 65°C with 0.6M NaCl and 80 μg proteinase K (Eurobio Scientific, GEXPRK01-I5). Samples were extracted once with Phenol – chloroform – isoamyl alcohol (Sigma, 77617), once with chloroform (Reactolab SA, P02410E16), ethanol precipitated and then resuspended in water. ChIP-enriched DNA were used to prepare libraries and processed with the Illumina TruSeq ChIP kit according to manufacturer specifications. Library molarity and quality were assessed with the Qubit (Thermofisher Scientific) and Tapestation (Agilent Technologies - DNA High sensitivity chip). Libraries were sequenced on a HiSeq 4000 and a NovaSeq 6000 Illumina sequencers for SR100 reads.