|
| Status |
Public on Sep 17, 2010 |
| Title |
MCF7-input-DNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
MCF7 cell line
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: MCF7
treatment (e=estrogen, n=control): N
chromatin ip antibody: total-DNA
chromatin ip antibody description: none
analysis batch: 17
|
| Treatment protocol |
The MCF7 cells were plated in 150 mm plates under normal growth conditions. When cell density reached 25% confluence, the media was changed to hormone free media for 96 h. The hormone deprived MCF7 cells were treated with ethanol or 20 nM estradiol (Sigma) for 6 h. For ChIP assays, the treated MCF7 cells were cross-linked with 1% formaldehyde for 15 min. The cross-linking reaction was stopped with 0.125 M glycine. The cross-linked cells were washed by PBS three times and stored at -80°C before use. The sonicated and fragmented, precleared chromatin lysate was incubated overnight with specific antibodies: ER (F-10, Santa Cruz), TRIM24 (Novus Biological), H3K4me2 (Active Motif) or normal sheep IgG (Upstate/Millipore).
|
| Growth protocol |
MCF7 cells were grown in estrogen-depeleted media for three days prior to time=0 of estrogen treatment.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the February 2009 human reference sequence (GRCh37) using the Illumina Genome Analyzer Pipeline using Illumina’s ELAND software.
Peaks: High-quality aligned read data is converted to the BED format and analyzed for peaks using the FindPeaks and the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) Peaks showing altered pull-down patterns are mapped to the genome, and a list of candidate genes and other known features flanking each peak are generated.
|
| |
|
| Submission date |
Sep 16, 2010 |
| Last update date |
May 15, 2019 |
| Contact name |
Bruce J Aronow |
| E-mail(s) |
bruce.aronow@chmcc.org
|
| Phone |
513-636-4865
|
| Organization name |
Cincinnati Children's Hospital Medical Center
|
| Street address |
|
| City |
Cincinnati |
| State/province |
OH |
| ZIP/Postal code |
45229 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (1) |
| GSE24166 |
Chromatin enrichment of TRIM24, estrogen receptor and H3K4me2 in estrogen-treated and -untreated MCF7 cells |
|
| Relations |
| SRA |
SRX026908 |
| BioSample |
SAMN00113435 |
