|
| Status |
Public on Dec 11, 2022 |
| Title |
MDA::ER::FOXA1_E2_2 |
| Sample type |
RNA |
| |
|
| Source name |
MDA::ER::FOXA1 cells treated for 4h with 10-8M estradiol
|
| Organism |
Homo sapiens |
| Characteristics |
gender: female
cell type: breast cancer cells
er status: Positive
integrated vectors: pCDNA3.1-Hygro-ER + pcDNA6/TR + pcDNA4/TO/FOXA1
|
| Treatment protocol |
Cells were plated at 2x106 cells per 9cmØ dishes in DMEM/2.5% FCS (complemented with 0.8 mg/ml hygromycin, 5 µg/ml Blasticidin and 75 µg/ml Zeocin) for 16h. Following two washes with PBS, cells were then cultivated for 3 days in DMEM/0.5% charcoal-stripped serum (BioWest) complemented with all antibiotics. Confluent cells were then treated with Estradiol (E2, Sigma) at a final concentration of 10-8 M for 4h or with equivalent amounts of ethanol vehicle (0.1%).
|
| Growth protocol |
Cells were maintained in DMEM (Sigma) supplemented with 5% fetal calf serum (FCS, BioWest) and antibiotics (Roche) at 37°C under 5% CO2. Media was complemented with 0.8 mg/ml Hygromycin (Calbiochem), 5 µg/ml Blasticidin (Invitrogen) and 75 µg/ml Zeocin (Invitrogen) for continuous selection of pCDNA3.1-Hygro-ER, pcDNA6/TR and empty pcDNA4/TO or pcDNA4/FOXA1 vectors, respectively.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini kit (Qiagen) with homogenization facilitated by QIAShredders (Qiagen). Integrity and purity of the RNAs were controlled on an Agilent Bioanalyzer using the RNA 6000 Nano Assay (Agilent). Ten micrograms of selected samples exhibiting a RIN >9.8 were then subjected to cDNA synthesis using the Superscript Double-Stranded cDNA Synthesis Kit (Invitrogen) and 50 pmol random hexamers. cDNAs were then treated for 10 min at 37°C with 5 μg RNaseA (Invitrogen), purified through a phenol:chloroform:isoamyl alcohol extraction on Phase Lock light gels (Fisher Scientific) and then precipitated.
|
| Label |
Cy3
|
| Label protocol |
Labeling was performed by NimbleGen Systems (Services, Reykjavic), following their standard operating protocol. See www.nimblegen.com.
|
| |
|
| Hybridization protocol |
Hybridization was performed by NimbleGen Systems (Services, Reykjavic), following their standard operating protocol. See www.nimblegen.com.
|
| Scan protocol |
Standard NimbleGen protocols (scanned at NimbleGen services, Reykjavic). See www.nimblegen.com.
|
| Description |
This sample is of MDA::ER::FOXA1 cells (MDA-MB231 cells engineered to constitutively express the estrogen receptor and to express FOXA1 in the presence of Tetracyclin) treated with 10-8M E2 for 4h. Second of the three independent replicates used in this experiment, each constituted by a pool of three separate cultures.
|
| Data processing |
Raw data (.pair) were subjected to the RMA algorithm (Robust Multi-Array Analysis; Irizarry et al. Biostatistics 4(2):249) with quantile settings (Bolstad et al. Bioinformatics 19(2):185) for background correction and normalization, as implemented within the ArrayStar software package (DNAstar, Inc.).
|
| |
|
| Submission date |
Mar 07, 2014 |
| Last update date |
Dec 11, 2022 |
| Contact name |
Raphaël Métivier |
| E-mail(s) |
raphael.metivier@univ-rennes1.fr
|
| Organization name |
CNRS UMR 6290
|
| Lab |
SP@RTE
|
| Street address |
Campus de Beaulieu
|
| City |
Rennes |
| ZIP/Postal code |
35042 Cedex |
| Country |
France |
| |
|
| Platform ID |
GPL15143 |
| Series (2) |
| GSE55680 |
Analysis of the impact of FOXA1 expression on MDA::ER E2-sensitive transcriptomes and ER cistromes [Transcriptome data] |
| GSE55741 |
Analysis of the impact of FOXA1 expression on MDA::ER E2-sensitive transcriptomes and ER cistromes |
|
