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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Mar 21, 2013 |
| Title |
Liver LCMRRBS 1 ng Replicate 1 |
| Sample type |
SRA |
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| Source name |
Liver LCMRRBS 1 ng Replicate 1
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| Organism |
Mus musculus |
| Characteristics |
method: LCM-RRBS
strain: C57BL/6
tissue: Liver
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Full details can be found in Schillebeeckx et al. (2013). Briefly, human tumor specimens were collected under an Institutional Review Board (IRB)-approved protocol. Immediately after surgery, a human endometrial tumor was divided in half. One half was fresh frozen while the other was formalin-fixed and paraffin-embedded (FFPE). 50 mg of fresh frozen endometrial tumor was cut into small pieces with a sterile scalpel blade and DNA extracted using the Maxwell 16 Tissue DNA Purification Kit (AS1030, Promega). The formalin-fixed paraffin-embedded preserved portion was cut into 6-µm sections onto microscope slides. Four 4 mm2 slices were scratched off the slide with a sterile scalpel blade, combined in 80 µl buffer and proteinase K (740901.50, Clontech), and incubated overnight at 65°C. Liver tissue was harvested from C57BL/6J mice and divided in half. One half was preserved in Tissue-tek optimal cutting temperature (O.C.T.) compound (25608-930, VWR) and snap frozen, while the other half was formalin-fixed and paraffin-embedded for downstream bulk DNA extraction and LCM. Fresh frozen, FFPE, and LCM samples were purified using NucleoSpin Tissue XS columns (740901.5, Clontech) following the protocol for laser-microdissected tissue and eluted in 20 μl of nuclease-free water.
Full details can be found in Schillebeeckx et al. (2013). For RRBS, genomic DNA was digested with MspI, size selected, end-repaired, 3'-terminal A extended and ligated to Illumina adapters containing methylated cytosines, bilsufite converted using the EZ DNA Methylation Gold Kit (ZYMO), PCR amplified with 18-24 cycles and sequenced. For LCM-RRBS , genomic DNA was digested with MspI, size selected, end-repaired, 3'-terminal A extended and ligated to Illumina adapters containing methylated cytosines, bilsufite converted using the EZ DNA Methylation Gold Kit (ZYMO), PCR amplified with 12 cycles and indexed primers, pooled, gel extracted, PCR amplifed with universal primers and 14 cycles, and sequenced
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Basecalls performed using CASAVA version 1.4
All analysis was performed using the February 2009 (GrCh37/hg19) build of the human genome and the July 2007 (NCBI37/mm9) build of the mouse genome. On average 25 million single-end 42-bp raw high quality reads per sample were either aligned to the cytosine-poor strand reference using the bisulfite mode of MAQ (41) or aligned to the reduced reference using RRBSMAP (42) filtering against reads that contain adapter sequence. Reads that showed less than 90% bisulfite conversion (~1 unconverted non-CpG cytosine per read) were filtered to remove those that resulted from incomplete bisulfite converted molecules. Aligned reads with a mapping quality of zero were also discarded. The resulting high quality uniquely mapped reads were used for methylation calling. We identified the genomic coordinates of all CpGs in the reference sequence and assessed percent DNA methylation by calculating the fraction of reads that had an unconverted cytosine at the CpG position relative to the total reads. We required that each read have either a “TG” or “CG” dinucleotide at the expected CpG coordinate to be considered for analysis.
Genome_build: mm9
Supplementary_files_format_and_content: bed tab-delimited txt files containing methylation calls for all CpGs
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| Submission date |
Mar 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Maximiliaan Schillebeeckx |
| E-mail(s) |
mschillebeeckx@wustl.edu
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| Phone |
314-362-1715
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| URL |
http://www.cgs.wustl.edu/~maxim
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| Organization name |
Washington University in St. Louis
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| Department |
Genetics
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| Lab |
Rob D. Mitra
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| Street address |
4444 Forest Parkway
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| City |
St. Louis |
| State/province |
MO |
| ZIP/Postal code |
63108 |
| Country |
USA |
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| Platform ID |
GPL13112 |
| Series (1) |
| GSE45361 |
Laser Capture Microdissection-Reduced Representation Bisulfite Sequencing (LCM-RRBS) maps changes in DNA methylation associated with gonadectomy-induced adrenocortical neoplasia in the mouse |
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| Relations |
| SRA |
SRX253001 |
| BioSample |
SAMN01985144 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1103091_Liver_LCMRRBS_FFPE_20mm_methcount.bed.gz |
124.6 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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