|
| Status |
Public on Oct 01, 2023 |
| Title |
Ctrl TGFb_R2 |
| Sample type |
RNA |
| |
|
| Source name |
HMECT cells
|
| Organism |
Homo sapiens |
| Characteristics |
source: Cell culture
transduction: control vector pWZL
treatment: TGFb
|
| Treatment protocol |
HMECT were transduced with a retrovirus pWZL control vector or pWZL RSK3 encoding vector. After one week of selection with neomycin, cells were treated or not with TGFβ (PeproTech) at the concentration of 0.5 ng/mL for 48h before RNA extraction.
|
| Growth protocol |
Immortalized human Mammary Epithelial Cells (HMECT) were grown in Mammary Epithelial Cell Growth Medium (MECGM, C-39115 Promocell) with 1% penicillin/streptomycin (Life Technologies). Cells were maintained at 37°C under a 5% CO2 atmosphere.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from HMECT cells in culture 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 |
1.65 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 Mouse GE 4x44K v2 Microarrays (Agilent-026655) 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 |
Sep 15, 2023 |
| Last update date |
Oct 01, 2023 |
| 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) |
| GSE243320 |
RSK3 switches cell fate: from stress-induced senescence to malignant progression |
|
