|
| Status |
Public on Feb 21, 2018 |
| Title |
HEC-TM-OHT96h-shSCN9A_R1 |
| Sample type |
RNA |
| |
|
| Source name |
Mammary epithelial cells immortalized with hTERT expressing anti SCN9A shRNA treated with 4-OHT during 96 hours
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: Human mammary epithelial cells (HECs; Lonza)
treatment: expressing anti SCN9A shRNA treated with 4-OHT during 96 hours
|
| Treatment protocol |
HECs were treated daily for 24 or 96 hours with 4-OHT (Sigma-Aldrich) at 250 nM, or with 65 mM KCl (Sigma-Aldrich),for 24 hours
|
| Growth protocol |
Human mammary epithelial cells (HECs; Lonza) were cultured in mammary epithelial cell growth medium (Promocell) with penicillin/ streptomycin 100 U/ml (Life Technologies). The cells were maintained in a humidified atmosphere at 37°C under 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from HEC-TM cells using NucleoSpin® RNA (Macherey Nalgen) following the manufacturer's recommendations
|
| Label |
Cy3
|
| Label protocol |
cRNAs were synthesized and labeled with the Cy3 dye from 100 ng of total RNA using the one-color Low Input Quick Amp Labeling Kit (Agilent Technologies) following the manufacturer's recommandations, followed by RNeasy Mini column purification (QIAGEN, Valencia, CA). Dye incorporation and cRNA yield were checked with the NanoDrop ND-1000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 ug of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. After fragmentation 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent GE Whole Human Genome Oligo Microarrays (Agilent-026652) for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried briefly in acetonitrile.
|
| Scan protocol |
Microarrays were scanned with an Agilent DNA microarray scanner G2565CA (Agilent Technologies).Fluorescent signals were extracted and normalized with the Feature Extraction software version 10.5.1.1 (Agilent Technologies),
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities.
|
| |
|
| Submission date |
Feb 20, 2018 |
| Last update date |
Feb 21, 2018 |
| Contact name |
jean-michel flaman |
| E-mail(s) |
jean-michel.flaman@lyon.unicancer.fr
|
| Phone |
+33687477812
|
| Organization name |
Inserm 1052
|
| Department |
CRCL
|
| Street address |
28 rue Laennec
|
| City |
Lyon |
| ZIP/Postal code |
69373 |
| Country |
France |
| |
|
| Platform ID |
GPL13497 |
| Series (1) |
| GSE110884 |
The SCN9A channel and plasma membrane depolarization promote cellular senescence through Rb pathway |
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