|
| Status |
Public on Apr 17, 2020 |
| Title |
DC2 cells treated with SAG, rep2 |
| Sample type |
RNA |
| |
|
| Source name |
DC2 immortalized epididymal principal murine cell line cultured in free serum
|
| Organism |
Mus musculus |
| Characteristics |
cell line: DC2
treatment: 250 nM of SAG
|
| Treatment protocol |
Immortalized DC2 cells were treated for 24 hrs with 250 nM of Smoothened agonist (SAG, n=3, SAG 1, SAG 2 and SAG 3), 20 mM of Cyclopamine (Cyclo, n=3, Cyclo 1, Cyclo 2 and Cyclo 3) and control medium condition (Ctrl, n=3, Ctrl 1, Ctrl 2 and Ctrl 3).
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| Growth protocol |
DC2 is a conditionally immortalized mouse epididymal epithelial cell line isolated from the distal caput of the mouse harbouring a temperature-sensitive simian virus 40 large T antigen. The functional expression of the SV40 large T antigen is induced by culturing the cells in vitro at permissive temperature (33°C). At a non-permissive temperature (37°C-39°C), the cells cease to proliferate.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from DC2 cells. Total RNA extraction was performed with the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol, and potential genomic DNA contamination was eliminated with the RNase-free DNase set (Qiagen). Total RNA was eluted in RNAse-free water and its concentration was quantified with a NanoDrop 1000 microvolume spectrophotometer (Thermo Scientific). Ribonucleic acid quality was assessed with the Eukaryote Total RNA Pico Series II kit (Agilent) on a 2100 Bioanalyzer by the transcriptomic core facility of the CHUQ. The minimal accepted number for RNA integrity was 7.
|
| Label |
Biotin
|
| Label protocol |
Total RNA (100 ng per sample) was labeled using the GeneChip® WT Plus Reagent Kit protocol and hybridized to the arrays as described by the manufacturer (Affymetrix, Thermofisher).
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| |
|
| Hybridization protocol |
Fifteen micrograms of fragmented cRNA were hybridized for 16 hrs at 45°C under constant rotation. After hybridization, chips were processed using the Affymetrix GeneChip Fluidic Station 450 (protocol EukGE-WS2v5_450). Staining was made with streptavidin-conjugated phycoerythrin (SAPE, Molecular Probes), followed by amplification with a biotinylated anti17 streptavidin antibody (Vector Laboratories), and followed by a second round of SAPE.
|
| Scan protocol |
The arrays were scanned using the Affymetrix GCS 3000 7G and the Gene-Chip Operating Software (Affymetrix, Santa Clara, CA), to produce the intensity files. Microarray hybridization was carried out at the Microarrays Facility of the Research Center of Laval University CRCHUL.
|
| Data processing |
Background subtraction and normalization of probe set intensities were performed using the method of Robust Multiarray Analysis (Irizarry et al, 2003), as impelemented by TAC 4.0 software..
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| |
|
| Submission date |
Jan 16, 2020 |
| Last update date |
Apr 18, 2020 |
| Contact name |
Ezequiel L Calvo |
| E-mail(s) |
cezequiel@yahoo.com
|
| Organization name |
CRCHUL
|
| Department |
Molecular Endocrinilogy
|
| Lab |
Microarrays
|
| Street address |
2705 Boul. Laurier
|
| City |
Quebec |
| State/province |
Quebec |
| ZIP/Postal code |
G1V 4G2 |
| Country |
Canada |
| |
|
| Platform ID |
GPL23038 |
| Series (1) |
| GSE143829 |
Hedgehog signaling pathway regulates gene expression profiling of epididymal principal cells through the primary cilium |
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