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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 08, 2013 |
| Title |
el018 |
| Sample type |
SRA |
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| Source name |
MCF-7_G-quadruplex pull-down
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| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7
cell type: Breast adenocarcinoma
enrichment method: G-quadruplex-specific probe
molecule subtype: genomic DNA enriched in DNA G-quadruplex structures
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| Growth protocol |
Human breast adenocarcinoma (MCF7) cells were obtained from the European Collection of Animal Cell Cultures and grown in Dulbecco’s Modified Eagle’s Media supplemented with 10% fetal calf serum (Sigma-Aldrich) at 37°C in 5% CO2.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was extracted from MCF7 cells using QIAamp DNA Mini Kit (Qiagen) according to the manufacturer’s protocol. The purified DNA was eluted from columns in 10 mM Tris pH 7.4. Genomic DNA was sheared by sonication sonicated with the Bioruptor™ UCD200 sonicator (Diagenode®) for 60 min at high power with a pulse of 30 s on/30 s off, to produce fragments of approximately 200 bp prior to pull-down experiment
50 μL of Protein A Dynabeads were washed three times with PBS then incubated with 2.5 μg of hf2 in 0.5 % BSA and 1 mg/mL yeast tRNA in PBS overnight rotating at 4 °C. Beads were washed three times with 0.5 % BSA then incubated with 400 μL of 300 ng/μL sonicated genomic DNA. Following overnight incubation rotating at 4°C, beads were washed 6 times with 10 mM Tris pH 7.4, 100 mM KCl, 0.1 % tween then once with 10 mM Tris pH 7.4, 100 mM KCl. Bound DNA was eluted in 50 μL of 1 % SDS, 0.1 M NaHCO3 at 30 °C for one hour then purified with Roche PCR purification columns. Recovered DNA was assessed for enrichment of telomeric sequences by qPCR with primers for telomeric repeats and a control genomic region in a regulatory region of the estrogen receptor gene without predicted G-quadruplex sequences. Recovered DNA was used to prepare libraries for Illumina sequencing using the TruSeq DNA library kit. Library quality was assessed using an Agilent Bioanalyzer 2100 before single end 36 base sequencing on the Illumina MiSeq, GAIIx or HiSeq 2000.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Description |
G-quadruplex pull-down
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| Data processing |
Basecalls performed using CASAVA 1.7 and OLB 1.9.4
Sequence reads were aligned to the Human Reference Genome (assembly hg18, NCBI Build 36.6, March 2008) using bwa 0.6.1 with default settings.
Reads mapped with MAPQ < 15 were filtered out. Reads overlapping regions producing aspecific binding were also removed (regions obtained from http://hgdownload-test.cse.ucsc.edu/goldenPath/hg18/encodeDCC/wgEncodeMapability/wgEncodeDukeRegionsExcluded.bed6.gz). Libraries were down-sampled to eight million reads using Picard/DownsampleSam (http://picard.sourceforge.net/command-line-overview.shtml#DownsampleSam).
Peak-calling was performed on the downsampled libraries using MACS 1.4 with default settings and the input library as control.
Genome_build: hg18
Supplementary_files_format_and_content: Text files produced by MACS reporting peak positons and statistics. Quoting the MACS manual (http://liulab.dfci.harvard.edu/MACS/00README.html): NAME_peaks.txt is a tabular file which contains information about called peaks. You can open it in excel and sort/filter using excel functions. Information include: chromosome name, start position of peak, end position of peak, length of peak region, peak summit position related to the start position of peak region, number of tags in peak region, -10*log10(pvalue) for the peak region (e.g. pvalue =1e-10, then this value should be 100), fold enrichment for this region against random Poisson distribution with local lambda, FDR in percentage. Coordinates in XLS is 1-based which is different with BED format.
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| Submission date |
Mar 18, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Dario Beraldi |
| E-mail(s) |
dario.beraldi@cruk.cam.ac.uk
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| Organization name |
Cambridge Research Institute
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| Street address |
Robinson Way
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| City |
Cambridge |
| ZIP/Postal code |
CB2 0RE |
| Country |
United Kingdom |
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| Platform ID |
GPL15520 |
| Series (1) |
| GSE45241 |
G-Quadruplex structures are stable and detectable in human genomic DNA. |
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| Relations |
| SRA |
SRX251931 |
| BioSample |
SAMN01983893 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1099727_el018.hf2_peaks.txt.gz |
68.4 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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