GSM1679450: WRN_ChIP_Control; Homo sapiens; ChIP-Seq
Pellets were washed once again with PBS-PMSF, re-centrifuged 1800 x g 10 minutes at 4oC and resupended in cell lysis buffer (5 mM PIPES, 85 mM KCl, 0.5% NP-40, 1 mM PMSF, supplemented with Protease Inhibitor Cocktail Tablets (Roche Applied Science, Indianapolis, IN, USA)). After 10 minutes incubation on a rotating device at 4oC, lysates were homogenized using a dounce homogenizer (25 strokes) to facilitate nuclei release. Nuclei were pelleted 1800 x g 10 minutes at 4C and incubated in nuclei lysis buffer (50 mM trishydroxymethylaminomethane (Tris) pH 8, 10 mM EDTA, 1% sodium dodecyl sulfate (SDS), 1 mM PMSF and supplemented with Protease Inhibitor Cocktail Tablets (Roche Applied Science, Indianapolis, IN, USA)) for 10 minutes on ice. Nuclear extracts were sonicated with a Bioruptor (Diagenode, Liege, Belgium) in order to obtain chromatin fragment of approximately 500 bp. Samples were centrifuged for 30 minutes at 16,000 x g in a tabletop centrifuge at 4C. The supernatant was collected and diluted in 2 volumes of dilution buffer (0.1 % SDS, 1% Triton X-100, 1.2 mM EDTA, 16.7 mM Tris pH 8, 167 mM NaCl, 1 mM PMSF supplemented with Protease Inhibitor Cocktail Tablets (Roche Applied Science, Indianapolis, IN, USA)). Samples were subjected to pre-clearing with Dynabeads® Protein G (Invitrogen, Burlington, ON, Canada) for 2 h on rotating device at 4oC, and incubated with 10 mg of either anti-WRN or an IgG non specific to WRN protein (as control) overnight on a rotating device at 4C. The control IgG was a rabbit polyclonal antibody raised against Fatty Acids Synthase (H-300) purchased from Santa Cruz biotechnologies (Santa Cruz, CA, USA). Dynabeads® Protein G were then added to the mix for 4h. Samples were washed twice with dialysis buffer (2 mM EDTA, 50 mM Tris pH 8, 0.2% SDS) and four times with wash buffer (100 mM Tris pH 9, 0.5 M LiCl, 0.2% NP-40, 1% sodium desoxycholate). After the last wash, protein complexes were eluted from beads with elution buffer (1% SDS, 50 mM NaHCO3), heated for 10 minutes at 65C and mix on a vortex at medium speed for 15 minutes. Elution was performed twice and both supernatants were pooled. Cross-links were reversed overnight at 65°C in the presence of 0.2 M NaCl, and the samples were then treated with RNase A and proteinase K. DNA was purified with standard phenol/chloroform and ethanol precipitation procedures. Quantification of precipitated DNA fragment was performed by qPCR using PerfeCTa® SYBR® Green SuperMix®, Low ROX™ according to the manufacturer’s instructions. ChIP DNA from the IgG control and WRN antibodies was used as the substrate for sequencing library preparation (TruSeq DNA sequencing kit [illumina, Valencia, CA]) per manufacturer instructions as previously described (Zheng et al. 2012).
GEO Accession
GSM1679450