|
| Status |
Public on Apr 27, 2012 |
| Title |
AR ChIP sequencing in 2hr 100nM DHT treated VCaP cells |
| Sample type |
SRA |
| |
|
| Source name |
VCaP prostate cancer cells, 2hr DHT treated, AR ChIP DNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: VCaP
cell type: prostate cancer cells
treatment: dihydrotestosterone (DHT)
treatment duration: 2hr
chip antibody: anti-AR
antibody vendor: Santa Cruz
antibody catalog number: sc-815x
antibody lot#: D1709
|
| Treatment protocol |
Hormone-depleted cells were treated with DHT to a final concentration of 100 nM. Cells treated with an equal volume vehicle, ETOH (ethanol) are used as controls
|
| Growth protocol |
The human prostate cancer VCaP cells (American Type Culture Collection) were maintained in DMEM supplemented with 10% fetal bovine serum (FBS), sodium pyruvate, sodium bicarbonate and penicillin:streptomycin solution at 37C under 5% CO2. For experiments requiring androgen/vehicle (DHT (Tokyo Chemical Industry) /EtOH) treatment, VCaP cells were grown in phenol red free DMEM supplemented with 10% charcoal stripped fetal bovine serum (CDFBS), sodium pyruvate, sodium bicarbonate and penicillin:streptomycin solution for 24 hours prior stimulation unless otherwise stated.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
All DNA samples were processed as per the Illumina Solexa ChIP-seq sample processing methods. VCaP cells were harvested, pelleted down, crosslinked and lysed with SDS (with protease inhibitor) before sonication was done. The sonicated chromatin was first decrosslinked and then purified by the QIAquick spin PCR purification kit (Qiagen, California) to obtain the genomic VCaP DNA for ChIP-Seq. 5 to 10 ng of ChIP DNA was end polished with T4 DNA polymerase and kinase. An 'A' base was added to the polished DNA fragments followed by the QIAquick column clean up. Solexa adaptors were ligated to the ChIP DNA fragments and enriched by 15 cycles of PCR amplification with Pfx DNA polymerase (Invitrogen) and Illumina primers. 200-300 bp size fractions were selectively isolated from the 2% ultra low range agarose gel and eluted by Qiagen gel extraction kit. The extracted DNA was quantified using Agilent bioanalyzer and subjected to Solexa sequencing according to the manufacturer's instructions.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
AR 2 hr
|
| Data processing |
Alignment: Sequence reads were obtained using the Illumina Genome Analyzer II Pipeline. In-house (Genome Institute of Singapore) BATMAN program was used for mapping the sequence tags to the reference genome hg18. In order to avoid potential PCR amplification bias, tags that shared the same mapping location on the same strand were removed. The uniquely-mapped reads with at most 2-mismatches were kept for further processing.
Peak calling: Binding peaks were determined using in-house Control based ChIPSeq Analysis Tools (CCAT) program with reference to a set of input reads as negative control. Peaks with a cut-off of FDR 0.05 were considered.
CCAT file format: chromosome, peakcenter, regionstart, regionend, tagcount, bgcount, zscore, fdr
|
| |
|
| Submission date |
Apr 28, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Kern Rei Chng |
| E-mail(s) |
[email protected]
|
| Organization name |
GIS
|
| Department |
Cancer Biology and Pharmacology 3
|
| Lab |
Cancer Biology and Pharmacology 3
|
| Street address |
60 Biopolis Street #02-01 Genome
|
| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE28950 |
TMPRSS2-ERG, HDACs and EZH2 are involved in an AR-centric transcriptional circuitry that calibrates androgenic response for prostate cancer progression (ChIP-Seq data) |
| GSE28951 |
TMPRSS2-ERG, HDACs and EZH2 are involved in an AR-centric transcriptional circuitry that calibrates androgenic response for prostate cancer progression |
|
| Relations |
| SRA |
SRX059704 |
| BioSample |
SAMN00262581 |
