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| Status |
Public on Dec 11, 2015 |
| Title |
HeLa E2F7_normoxia |
| Sample type |
SRA |
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| Source name |
HeLa_normoxia
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HeLa
antibody: E2F7 (sc-66870, Santa Cruz Biotechnology)
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| Growth protocol |
The cervical cancer (HeLa) cell line was cultured in DMEM (Invitrogen, 41966-052) supplemented with 10% FBS (Lonza, DE14-802F) and 1% Penicillin/Streptomycin (Lonza, DE17-602E).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP was performed according the EZ ChIP protocol (Upstate, 17-371) with the following specifications: HeLa cells were seeded at day 1 at a concentration of 7x10^6 cells per 145mm plate. Next day, cells were cultured overnight in hypoxia, or continued to grow under normoxia, and harvested on day 3. For single and double ChIP, 5 145mm plates were used per condition. In vitro crosslinking was performed on a shaker at room temperature (RT) for 10 minutes, using 1% freshly made paraformaldehyde. Crosslinking was quenched for 5 minutes incubation at RT on a shaker. Next, cells were washed twice on ice with PBS (40C, supplemented with protease inhibitors (11873580001, Roche)), and harvested, pelleted and resuspended in 2ml lysis buffer (0.3% SDS, 10mM EDTA, 50mM Tris and protease inhibitors). Sonication (10 cycles of 10 seconds followed by 1 minute incubation on ice) was performed in a FACS tubes using a Soniprep 150 sonicator (MSE). 10µl of sonicated sample was analyzed on gel to check for sonication efficiency. Sonicated lysates were centrifugated (10 minutes, 40C) to remove insoluble components and large DNA fragments. 200µL lysate was used per ChIP sample. The DNA concentration in the sonicated lysate was measure using a Nanodrop to normalize the amount of input DNA between normoxic and hypoxic ChIP samples. Protein G agarose beads (16-266, Milipore) were coated overnight in 0,1% BSA (Sigma, A3294), and 60 µL was used for pre-clear (2h, 40C) and final precipitation of immune complexes (1h, 40C). For single ChIP 5µg and for double ChIP 10g was used. immunorecipitation were performed overnight at 40C on a rotating platform. In case of double ChIP, eluates were diluted in dilution buffer and incubated with another 10ug of antibody overnight. The following antibodies were used: ChIP grade HIF1α (ab2185, Abcam) E2F7 (sc-66870), E2F1 (sc-193), IgG (2729S; cell signaling). De-crosslinked DNA was purified over a column (Qiagen, 28106) and eluted in 65 µl H2O
immunoprecipitated chromatin was sheared, end-repaired, sequencing adaptors were ligated and the library was amplified by ligation mediated PCR (LMPCR). After LMPCR, the library was purified and checked for the proper size range and for the absence of adaptor dimers on a 2% agarose gel, barcoded and sequenced on SOLiD/AB sequencer in multiplexed way to produce 50-bp long reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
AB SOLiD 4 System |
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| Description |
ChIP DNA
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| Data processing |
Sequencing reads were mapped against the reference genome (hg19 assembly, NCBI build 37) using BWA package
Multiple reads mapping to same location and strand have been collapsed to single read and only uniquely placed reads were used for peak-calling. Cisgenome was used for peak-calling from the ChIP-seq data.
Genome_build: hg19
Supplementary_files_format_and_content: peak coordinates file : number_chromosome_start_end_length_fold-enrichment
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| Submission date |
Mar 16, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Michal Mokry |
| E-mail(s) |
m.mokry@umcutrecht.nl
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| Organization name |
Wilhelmina Children's Hospital, University Medical Center Utrecht
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| Street address |
Lundlaan 6
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| City |
Utrecht |
| ZIP/Postal code |
3584 EA |
| Country |
Netherlands |
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| Platform ID |
GPL13393 |
| Series (1) |
| GSE66956 |
Genome-wide analysis reveals NRP1 as a critical HIF1-E2F7 target gene in the regulation of motorneuron guidance in vivo |
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| Relations |
| BioSample |
SAMN03418618 |
| SRA |
SRX957673 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1634923_ne2f7_20110913.unique_peak.cod.gz |
57.9 Kb |
(ftp)(http) |
COD |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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