|
| Status |
Public on Dec 04, 2010 |
| Title |
[E-MTAB-223] T47D_input_lane2 |
| Sample type |
SRA |
| |
|
| Source name |
T47D_Input_CRI02
|
| Organism |
Homo sapiens |
| Characteristics |
material type: cell_line
cell line: T-47D
treatment: doxycycline
timepoint: 48h
Sex: female
disease state: breast cancer
chip antibody: input_DNA
|
| Treatment protocol |
Drug treatments were performed with Dox (Clontech) at 0.1 ug/ml, R5020 (Du Pont) at 10 nM or 17beta-estradiol (Sigma) at 10 mM. Dox-containing medium was changed every 24 hours to ensure optimum Dox activity. Adherent cells on tissue culture plates were incubated in fresh, warm, serum-free medium supplemented with 1% formaldehyde, at 37 C for 10 minutes. Cells were washed 2 times with cold PBS and scraped into 600 ul PBS with protease inhibitors (P8340, Sigma). Fixed cells were spun for 2 minutes at 6,000xg, washed as before and snap frozen in liquid nitrogen.
|
| Growth protocol |
grow | RPMI 1640 medium por DMEM supplemented with 10% inactivated FBS, l-glutamine and PEST at 37C with 5% CO2.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
immunoprecipitate | Antibodies were from Santa Cruz Biotechnology (sc-543), abcam (ab5089 and ab23738) and Millipore (07-729) and ChIP was performed using 10e7-10e8 cells per reaction. Briefly, crosslinking of protein and DNA was done in room temperature for 10 minutes with serum free media containing 0,37 % formaldehyde. Cells were scraped and washed in PBS before treatment of cell lysis buffer with protease inhibitors on ice. Pelleted nuclei were resuspeded in RIPA buffer and a BioRuptor (Diagenode) was used to sonicate DNA. ChIP was performed with 10 micrograms of antibody. ChIP DNA was gel fractionated and amplified to create two size libraries corresponding to insert sizes of 200-300 bp. For DNase I hypersensitivity was performed as previously described (Eeckhoute et al., 2006). Digested DNA was run on a gel and the digested material (between 200bp and 500bp in size). For all ChIPs washing was done six times with RIPA buffer and once with TE-buffer and DNA-protein complexes were eluted from beads twice with 200 microlitres of 0.1 M NaHCO3 and 1 % SDS and treated with RNAseA at 65C for 6 hours and Proteinase K at 45C over night. Chromatin was amplified with Solexa protocol chromatin amplification.
sequencing | Sequencing was carried out by the genomic service of Cambridge Research Institute-CRUK using the Illumina genome analyzer II
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Performer: CRUK-CRI
|
| Data processing |
Sequences generated by the Illumina genome analyzer were aligned against NCBI Build 36.3 of the human genome using MAQ with default parameters
Uniquely aligning reads were converted to BED format using a custom script.
Peaks were called using MACS (version 1.4.0alpha2; Zhang et al, 2008) using only uniqely aligned reads
scanning | The Eland software (Illumina) was used to align reads against the hg18 reference genome. The first 32 bases were aligned allowing a maximum of 2 mismatches. Reads aligning to multiple locations were discarded. The remaining un-aligned reads were shortened by 3 bases at the 3' end and the procedure repeated until the remaining aligned sequences were 23 bases long.
|
| |
|
| Submission date |
Nov 30, 2010 |
| Last update date |
May 15, 2019 |
| Organization |
European Bioinformatics Institute |
| E-mail(s) |
miamexpress@ebi.ac.uk
|
| Lab |
ArrayExpress
|
| Street address |
Wellcome Trust Genome Campus
|
| City |
Hinxton |
| State/province |
Cambridgeshire |
| ZIP/Postal code |
CB10 1SD |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL10999 |
| Series (1) |
| GSE25710 |
[E-MTAB-223] ChIP-seq for FOXA1, ER and CTCF in breast cancer cell lines |
|
| Relations |
| SRA |
ERX008609 |
