|
| Status |
Public on Sep 14, 2016 |
| Title |
786O_2 |
| Sample type |
SRA |
| |
|
| Source name |
Renal Carcinoma Cell Line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: 786-O
treatment: Normoxia
cell line modification: none
|
| 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 2500 |
| |
|
| 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 |
GPL16791 |
| 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 |
SAMN04503282 |
| SRA |
SRX1592834 |
