|
| Status |
Public on Apr 22, 2021 |
| Title |
EyeBank_cultured_corneal_endothelial_cells_001_without TGF-beta2_rep1 |
| Sample type |
RNA |
| |
|
| Source name |
primary corneal endothelial cell population
|
| Organism |
Homo sapiens |
| Characteristics |
disease: no corneal pathology associated
Sex: M
age: 72
treatment: control
cell type: primary corneal endothelial cell population
|
| Treatment protocol |
At confluency, culture medium was switched for OptiMem-I, fetal bovin serum and penicilin/streptomycin supplemented or not with TGF-beta2 for 7 days.
|
| Growth protocol |
[Isolation protocol] Cells were isolated from mechanically peeled Descemet's membrane using collagenase A (1mg/ml) treatment. They were then seeded in culture dishes for expansion.
Cell populations were expanded in culture medium made of OptiMem-I, 8 % fetal bovine serum, 5 ng/mL human epidermal growth factor, 20 μg/mL ascorbic acid, 0.08 % chondroitin sulfate and a 1X mix of penicilin and streptomycin. Cells were maintained in 37°C and 8% CO2 incubators.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using the RNeasy Mini Kit (QIAGEN, Cat#74104)
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 50-200 ng RNA using the One-Color LowInput QuickAmp Labeling Kit One-Color (Agilent) according to the manufacturer's instructions
|
| |
|
| Hybridization protocol |
600 ng of Cy3-labelled cRNA (specific activity >8.0 pmol Cy3/ug cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 ul containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 25 ul of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole SurePrint G3 Human GE 8x60K (G4851A) 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 immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the Agilent SureScanner (G4900DA) using one color scan setting for 1x60k array slides (Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
Gene expression of passage 3 cells after 7 days post-confluency
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7 (Agilent) using default parameters to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded.
Supplementary file processed_data.txt forms the basis of conclusions in the manuscript.
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| |
|
| Submission date |
Apr 21, 2021 |
| Last update date |
Apr 22, 2021 |
| Contact name |
Stéphanie Proulx |
| Organization name |
Université Laval
|
| Street address |
1050 Chemin Ste-Foy
|
| City |
Québec |
| State/province |
Qc |
| ZIP/Postal code |
G1S4L8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE172573 |
TGF-β modulation of cell-cell and cell-extracellular matrix interactions in post-confluent corneal endothelial cells |
|
