|
| Status |
Public on Dec 06, 2012 |
| Title |
Pat45_MeDIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
prostate tumor biopsy
|
| Organism |
Homo sapiens |
| Characteristics |
tumor stage: pT3
gleason: 4
|
| Growth protocol |
Primary samples from 51 prostate cancer tissues were obtained after radical prostatectomies.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA preparation: For Laser Capture Microdissection (LCM, Zeiss, Germany) of epithelial cells, 16µm tissue sections were mounted on special LCM slides and briefly stained with hematoxilin and eosin to facilitate localization of epithelial cells. Epithelial cells were collected by LCM from 10 tissue sections each. DNA was isolated using the DNA mini kit (Qiagen) according to the manufacturer's instructions. From macrodissected samples the AllPrep DNA/RNA/Protein Mini Kit was used.
Methylation profiling by MeDIP-Seq: In brief, 2.5µg of genomic DNA were fragmented to 100 to 200bp using the Covaris S2 system and end repaired with End Repair mix (Enzymatics) followed by a purification step (Qiagen DNA purification kit). Barcoded sequencing adapters were ligated followed by nick translation with DNA polymerase I (NEB, 10U). For the enrichment step of the methylated DNA immunoprecipitation (MeDIP) 5µg of an anti-5-methyl cytosine antibody (Eurogentec) coupled to magnetic beads were used. Coupling was performed by incubation overnight in 1xPBS + 0.5% BSA. Sequencing libraries were generated prior to the enrichment and incubated with the beads for 4 hours in IP Buffer (10mM sodium phosphate buffer pH 7, 140mM NaCl, 0.25% Triton X100). Beads were washed three times with IP buffer and DNA was eluted in elution buffer (50 mM Tris-HCl pH 7.5, 10 mM EDTA, 1% SDS) by incubation for 15min at 65°C. After two hours of incubation with proteinase K, the DNA was phenol/chloroform extracted and ammonium acetate/ethanol precipitated. Enrichment controls were performed with real time PCRs targeting methylated as well as unmethylated regions. SOLiD sequencing libraries were prepared following the SOLiD v3 fragment multiplex library preparation protocol (Life Technologies) with slight modifications. Following MeDIP enrichments libraries were amplified with multiplex library PCR primers 1 and 2, size-selected and quantified. Sequencing was performed on a SOLiD 3+ using barcode sequencing chemistry (5+35 bp) (Lifetech).
Barcoded sequencing libraries were prepared according to the SOLiD protocol , enriched with an anti-5methyl cytosine antibody (Eurogentec: Clone 33D3) and sequenced with the SOLiD fragment 35+5bp barcode sequencing technology (Lifetech).
|
| |
|
| Library strategy |
MeDIP-Seq |
| Library source |
genomic |
| Library selection |
5-methylcytidine antibody |
| Instrument model |
AB SOLiD System 3.0 |
| |
|
| Description |
IP against methylated Cytosine
|
| Data processing |
Bed files: Reads were aligned to HG19 using the Bioscope v1.0 pipeline from Lifetech with standard settings for 35mer reads. The aligned reads were elongated to 200bp in a strand-oriented manner. Redundant reads and reads with no CpGs in the elongated sequence were excluded.
Genome Build:
Pat45_MeDIP-seq_sorted_filtered.bed.gz: GRCh37
|
| |
|
| Submission date |
Jan 25, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Michal-Ruth Schweiger |
| E-mail(s) |
mschweig@molgen.mpg.de
|
| URL |
http://www.molgen.mpg.de/~cg/
|
| Organization name |
Max-Planck-Institut for Molecular Genetics
|
| Department |
Vertebrate Genomics
|
| Lab |
Cancer Genomics
|
| Street address |
Ihnestr. 63-73
|
| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
| |
|
| Platform ID |
GPL9442 |
| Series (1) |
| GSE35342 |
Genome-wide DNA methylation events in TMPRSS2:ERG fusion negative prostate cancers implicate an EZH2 dependent mechanism with miRNA-26a hypermethylation. |
|
| Relations |
| SRA |
SRX117863 |
| BioSample |
SAMN00779091 |
