|
| Status |
Public on Feb 10, 2012 |
| Title |
MCF-7,LTED_ER-ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
MCF-7/LTED cells
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: MCF-7
chip antibody: Mouse-anti-ER*
vendor: Neomarkers, clone Ab-10 (TE111.5D11)
catalog #: MS-315-P
lot #: 315P508A
chip antibody: Rabbit-anti-ER*
Santa Cruz Biotechnology, clone HC-20
catalog #: SC-543
lot #: C2510
|
| Treatment protocol |
Cells were treated with IMEM + 10% DCC-FBS (no phenol red)
|
| Growth protocol |
Cells were maintained in IMEM + 10% DCC-FBS (no phenol red)
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and ER-DNA complexes were isolated with antibody. Thirty nanograms of ChIP DNA was end-polished with T4 DNA polymerase and kinase. An adenosine base was added to the polished DNA fragments followed by Qiaquick column clean-up. Solexa adaptors were ligated to the ChIP DNA fragments. Samples were run on a 2% e-gel (Invitrogen) for 10 min. A gel region from 200-400 bp was excised (average 300 bp), and DNA was purified using Gel Extraction Kit (Qiagen). Samples were amplified by PCR using 2X Phusion Master Mix and Paired End Primers 1.0 and 2.0 for 18 cycles. Samples were purified using Nucleotide Extraction Kit (Qiagen) and eluted in 12 mL EB Buffer. Samples were then run on a DNA-1000 chip to check the average size of the library prep. All samples had an average size of 300 bp. Library preps were next subjected to Solexa sequencing using the Genome Analyzer II according to the manufacturer’s instruction.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Chromatin IP against ERa
|
| Data processing |
Alignment: Sequence reads were obtained and mapped to the human (March, 2006) genomes using the Illumina Genome Analyzer Pipeline. All reads mapping with two or fewer mismatches were retained and read starts were summed in sliding windows of 400 bp to create summary windows.
Peaks: Peak detection was performed with the Model-based Analysis of ChIP-Seq (MACS) algorithm (http://liulab.dfci.harvard.edu/MACS/) (Zhang et al., 2008) in Python with the following parameters: effective genome size = 2.7 x 109; band width = 300; model fold = 10,30; p-value cutoff = 10-5; range for calculating regional lambda = 10,000 bp; tag size = 43 bp; maximum duplicate tags at the same position in treatment = 2.
|
| |
|
| Submission date |
Feb 14, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Todd W Miller |
| E-mail(s) |
todd.miller@vanderbilt.edu
|
| Organization name |
Vanderbilt Univ
|
| Department |
Medicine
|
| Street address |
2220 Pierce Ave, 777 PRB
|
| City |
Nashville |
| State/province |
TN |
| ZIP/Postal code |
37174 |
| Country |
USA |
| |
|
| Platform ID |
GPL9115 |
| Series (2) |
| GSE27300 |
Estrogen-independent genomic ER binding analysis |
| GSE37955 |
ERa-dependent E2F transcription can mediate resistance to estrogen deprivation in human breast cancer |
|
| Relations |
| SRA |
SRX042342 |
| BioSample |
SAMN00210996 |
