GSM1679450: WRN ChIP Control; Homo sapiens; ChIP-Seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
TFs and others
Antigen
WRN
Cell type
Cell type Class
Kidney
Cell type
293
Primary Tissue
Kidney
Tissue Diagnosis
Normal
Attributes by original data submitter
Sample
source_name
HEK293_WRN_CHIP
cell line
HEK293
cell type
Human 293 embryonic kidney cells
crosslinker
formaldehyde
antibody used
IgG non specific antibody
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
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).