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| Status |
Public on May 09, 2011 |
| Title |
PrEC-1 |
| Sample type |
SRA |
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| Source name |
PrEC
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| Organism |
Homo sapiens |
| Characteristics |
tissue: Prostate
cell line: PrEC
cell type: Normal Cell Line
origin: Cell Line
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| Growth protocol |
Human primary prostate epithelial cells were purchased from Lonza Inc. (Mapleton IL.), and the prostate cancer cell line LNCaP was obtained from ATCC (Manassas, VA). The PrEC and LNCaP cells were grown in PrEGM media (Lonza Inc. Mapleton IL) and RPMI 1640 containing 10% FBS (Invitrogen, Carlsbad, CA), respectively.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Methylplex library synthesis and GC-enrichment was obtained through a commercial service at Rubicon Genomics Inc. Ann Arbor, MI. Briefly, fifty nanograms of gDNA from tissues or cells were digested with a proprietary cocktail of methylation-sensitive restriction enzymes and then amplified by PCR with universal primers to create a MethylPlex library that is enriched for methylated DNA. MethylPlex DNA was then subjected to additional enzymatic treatment to deplete all non-GC-rich DNA sequences, purified and amplified in a second round of PCR. This created a highly enriched library of fully methylated GC-rich regions of the human genome, representing about 1% of total DNA. After purification the amplification adaptors were removed by a restriction enzyme digestion. One and five micrograms of the purified products from each cell line were directly incorporated into the genomic DNA sequencing sample preparation kit procedure of Illumina (Illumina Inc, San Diego, CA) at the end repair step, skipping the nebulization process. An adenine base was then added to the purified end repaired products using Klenow exo (3’ to 5’ exo minus) enzyme. The reaction product was purified, ligated to illumina adaptors with DNA ligase and resolved on a 2% agarose gel. For LnCaP and PrEC libraries gel pieces were excised at 200 and 400 base pair positions and the DNA was extracted using Qiagen gel extraction kit (Qiagen Inc, Valencia, CA). Subsequently for all tissue samples 400bp gel cut was utilized. One ul of this eluate was used as a template in a PCR amplification reaction with Phusion DNA polymerase (Finnzymes, INC., Woburn, MA) to enrich the adapter modified DNA fragments. The PCR product was purified and analyzed by Bioanalyzer (Agilent Technologies, San Diego, CA) before using it for flow cell generation, where 10nM of library was used to prepare flowcells with approximately 30,000 clusters per lane.
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| Library strategy |
MRE-Seq |
| Library source |
genomic |
| Library selection |
Restriction Digest |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
CpG enriched DNA library
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| Data processing |
HPEAK: A Hidden Markov Model (HMM)-based algorithm previously used for Chip-Seq data analysis(http://www.sph.umich.edu/csg/qin/HPeak) was used to locate peaks from mapped reads obtained in each sequencing run.
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| Submission date |
Mar 01, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Jung Kim |
| E-mail(s) |
[email protected]
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| Organization name |
University of Michigan
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| Street address |
1400 E. Medical Center Drive
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| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
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| Platform ID |
GPL9115 |
| Series (2) |
| GSE27618 |
DNA methylation analysis of prostate cancer cell lines and tissues using Next Generation Sequencing |
| GSE27619 |
Deep sequencing reveals distinct patterns of DNA methylation and transcript isoform regulation in prostate cancer |
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| Relations |
| SRA |
SRX062041 |
| BioSample |
SAMN02061454 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM684593_20FAMAAXX_1_PrEC.allregions.txt.gz |
699.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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