|
| Status |
Public on Jun 03, 2011 |
| Title |
AP-2γ ChIP sequencing in ETOH (ethanol) treated MCF-7 cells |
| Sample type |
SRA |
| |
|
| Source name |
MCF-7 breast cancer cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF-7 breast cancer cell line
agent: ethanol
antibody: anti-AP-2γ
antibody vendor: Santa Cruz
antibody batch/lot#: G1404
antibody catalog#: sc-8977 (H-77)
|
| Treatment protocol |
Hormone-depleted cells were treated with E2 to a final concentration of 100 nM for 45 mins before the ChIP procedure. Cells treated with an equal volume vehicle, ETOH (ethanol) for 45 min are used as controls
|
| Growth protocol |
MCF-7 breast cancer cells were maintained in DMEM (Invitrogen/Gibco) supplemented with 5% fetal bovine serum (FBS), penicillin, streptomycin, and gentamycin in a 37°C incubator with 5% CO2. Prior to 17 beta-estradiol (“estrogen/E2”, Sigma) stimulation, ~4 millions cells were seeded into 150mm dish and starved with phenol red-free DMEM/F12 containing 5% charcoal stripped FBS (Hyclone) with penicillin, streptomycin and gentamycin for 96 hours to 80% confluence.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
All DNA samples were processed as per the Illumina Solexa ChIP-seq sample processing methods. 5 to10 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 instruction.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
AP-2γ ChIP sequencing, after ETOH (ethanol) treatment
|
| 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 stringent cut-off of FDR 0.005 were considered. See 'significant.peak' files.
CCAT file format: chromosome, peakcenter, regionstart, regionend, tagcount, bgcount, zscore, fdr
|
| |
|
| Submission date |
Aug 30, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
SI KEE TAN |
| E-mail(s) |
tansk99@gis.a-star.edu.sg
|
| Organization name |
Genomic Institute of Singapore
|
| Department |
Cancer biology and Pharmacology
|
| Street address |
60 Biopolis Street #02-01 Genome
|
| City |
Singapore |
| ZIP/Postal code |
Singapore 138672 |
| Country |
Singapore |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE23852 |
Activator protein-2γ is a critical determinant of estrogen receptor interactome formation and gene transcription in breast cancer [ChIP-Seq] |
| GSE26741 |
Activator protein-2γ is a critical determinant of estrogen receptor interactome formation and gene transcription in breast cancer |
|
| Relations |
| SRA |
SRX028632 |
| BioSample |
SAMN00115794 |
