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Sample GSM2067357 Query DataSets for GSM2067357
Status Public on Sep 14, 2016
Title 786O_VHL_N
Sample type SRA
 
Source name Renal Carcinoma Cell Line
Organism Homo sapiens
Characteristics cell line: 786-O+VHL
treatment: Normoxia
cell line modification: stable WT VHL expression
Treatment protocol For hypoxia treatment cells, were incubated for 16 hr in an In Vivo2 Hypoxia Work station (Ruskinn Technology) at 0.5% oxygen.
Growth protocol Dulbecco Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 100U/ml penicillin and 100 μg/ml streptomycin.
Extracted molecule genomic DNA
Extraction protocol Capture-C libraries were prepared as previously described (Davies et al. 2016 Nature Methods). Cells were fixed with 2% (vol/vol) formaldehyde for 10 min, quenched with 125 mM glycine in PBS and then lysed in cold lysis buffer (10 mM Tris-HCl, pH 8, 10 mM NaCl, 0.2% Igepal, 1× cOmplete Protease Inhibitor Cocktail (Roche). Chromatin was digested with Dpn2 (New England Biolabs) at 37oC overnight. Fragments were then diluted and ligated with T4 DNA ligase (Thermo Scientific) at 16oC overnight. Crosslinking was reversed by overnight incubation at 60oC with proteinase K (Bioline). The 3C libraries were then purified by phenol-chloroform and chloroform extraction followed by precipitation in ethanol at -80oC overnight. Digestion efficiency was determined by qPCR and gel electrophoresis. Sequencing libraries were prepared from 5 μg of each 3C library by sonication using a S220 focused-ultrasonicator (Covaris) to a average size of 200bp and indexed using NEBnext reagents (New England Bioloabs) according to protocol. Enrichment of 1-2 μg of indexed library incubated with 13 pmol of a pool of biotinylated oligonucleotides (Intergrated DNA technologies or Sigma) was performed using the SeqCap EZ system (#06953212001, Roche/Nimblegen) following the manufacturer’s instructions. Two rounds of capture employing 48-72 hr and 24 hr hybridizations respectively were used. Capture enrichment was determined by qPCR. Correct library size was confirmed using a Bioanalyzer DNA 1000 or Tapestation D1000 kit (Agilent), and DNA concentrations were determined using a Qubit 2.0 Fluorometer (ThermoFisher Scientific).
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 4000
 
Data processing Reads were trimmed using trim galore and then in silico digested with DpnII using dpnII2E.pl (https://github.com/telenius/captureC/releases)
Resulting reads were then algined to genome using bowtie 1.0.0
Interaction frequencies were then determined using CCanalyser2.pl (https://github.com/telenius/captureC/releases)
Genome_build: hg19
Supplementary_files_format_and_content: bigwig files contain interaction frequency from all sites
 
Submission date Feb 19, 2016
Last update date May 15, 2019
Contact name James Platt
Organization name University of Oxford
Street address Roosevelt Drive
City Oxfors
ZIP/Postal code OX3 7BN
Country United Kingdom
 
Platform ID GPL20301
Series (2)
GSE78100 Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters
GSE78114 Capture-C reveals preformed chromatin interactions between HIF-binding sites and distant promoters.
Relations
BioSample SAMN04503279
SRA SRX1592839

Supplementary file Size Download File type/resource
GSM2067357_786OVHL_N.bw 2.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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