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| Status |
Public on Jun 19, 2017 |
| Title |
MCF7_LTED.RE.slide1 |
| Sample type |
SRA |
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| Source name |
Breast cancer cell line
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| Organism |
Homo sapiens |
| Characteristics |
cell type: long-term estradiol deprived (LTED) MCF-7 cells
genomic dna digested with: methylation-sensitive restriction endonucleases (RE)
genomic dna fraction: methylated
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| Growth protocol |
MCF7 LTED cells were maintained in RPMI 1640 without phenol red supplemented with 5% charcoal stripped fetal bovine serum, 10 mM HEPES, 4.5 g/L glucose, 2 mM L-glutamine, 1 mM sodium pyruvate, and 50 µg/ml gentamicin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated from 3 x 10^6 cells using the Quick-gDNA MiniPrep kit from Zymo Research (Irvine, CA) according to the manufacturers instructions. The quality of the isolated high molecular weight DNA was confirmed by agarose gel electrophoresis.
Methyl-MAPS libraries were prepared according to the protocol in Edwards et al Genome Research 2010. In brief, genomic DNA was split and unmethylated and methylated compartments were independently obtained by limit digestions of 10–15 ug of DNA with McrBC or five tetranucleotide methylation-sensitive restriction endonucleases (RE; HpaII, HhaI, AciI, BstUI, HpyCH4IV). Digested fragments were size selected by gel-electrophoresis for fragments >800 bp. Mate-pair SOLiD sequencing libraries were then prepared using custom 6 bp barcodes. As part of the Methyl-MAPS protocol, EcoP15I CAP linkers are ligated onto the ends of the fragments. The fragments are then circularized with biotinylated internal adapters, and digested with EcoP15I, which cleaves the DNA 25-27 bp away from its recognition site. Consequently, although we sequence 35 bp for both the F3 and R3 tags, only 25 bp comprise the genomic sequence tag. Each library was loaded on two sequencing slides.
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| Library strategy |
MRE-Seq |
| Library source |
genomic |
| Library selection |
Restriction Digest |
| Instrument model |
AB SOLiD System 3.0 |
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| Data processing |
Library strategy: Methyl-MAPS (Methylation Mapping Analysis by Paired-end Sequencing)
Tags for each sample and library (methylated and unmethylated compartment) were sequenced on two slides along with other samples. Tags were distinguished using custom barcodes (6 bp in length) on the F3 tags. Errors in the barcodes were recovered up to 2 bp (in colorspace). Tags were then trimmed to 25 bp.
ag mapping was performed using Corona Lite from the Applied Biosystems SOLiD System software analysis package. Paired tags were mapped individually in color space, allowing up to two mismatches in each 25-bp tag to hg18. Up to 10 hits per chromosome were recorded for each tag.
F3 and R3 tags were paired using the Corona Lite package. A mate pair was reported if a single uniquely placed pair could be made from the hits of each tag that met the contraints of order, orientation, and distance (0-15 kb). If no mate pair could be made, then mate-pair rescue was attempted in which each hit was used as an anchor and the region where an appropriate mate should be found was scanned while allowing a total of four mismatches across both tags. A rescued mate pair was reported if a single uniquely placed pair could be made.
Mapped reads from the same sample were pooled across slides. Fragment filtering, fragment count normalization and methylation level calculation was performed as in Edwards et al. 2010 Genome Research.
Genome_build: hg18
Supplementary_files_format_and_content: SOLiD .mates file; 4-column tab-delimited (chromosome, position, value, coverage)
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| Submission date |
Nov 12, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
John R. Edwards |
| E-mail(s) |
jredwards@wustl.edu
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| Organization name |
Washington University School of Medicine
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| Department |
Center for Pharmacogenomics
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| Street address |
660 S. Euclid Ave, Campus Box 8220
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63110 |
| Country |
USA |
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| Platform ID |
GPL9442 |
| Series (2) |
| GSE74942 |
Epigenetic activation of the prostaglandin receptor EP4 promotes resistance to endocrine therapy for breast cancer [Methyl-MAPS] |
| GSE74943 |
Epigenetic activation of the prostaglandin receptor EP4 promotes resistance to endocrine therapy for breast cancer |
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| Relations |
| BioSample |
SAMN04262304 |
| SRA |
SRX1427946 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1938086_MCF7_LTED.RE.slide1.mates.txt.gz |
687.5 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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