|
| Status |
Public on Dec 22, 2014 |
| Title |
hu-beta-celline_siRFX6_rep3 |
| Sample type |
RNA |
| |
|
| Source name |
hu-beta-celline_siRFX6_rep3
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: EndoC-βH2 cells
|
| Treatment protocol |
For siRNA-based gene knockdown studies, EndoC-βH2 cells were transfected using Lipofectamin RNAiMAX (life technologies) following manufacturer’s instructions with minor modifications. Briefly, freshly trypsinized EndoC-βH2 cell suspension (5x104 cells/cm2) were incubated with lipofectamin-siRNA complex in Opti-MEM containing 2-mercaptoethanol, nicotinamide, transferrin and selenite for 3-4 minutes and next plated. 3-5h later, the medium was replaced. ON-TARGETplus siRNA SMARTpool for human RFX6 gene (~30 nM) and ON-TARGETplus Non-targeting pool (siNT) (Dharmacon, Thermo Scientific) were used. Cells were harvested 72 h post transfection for further analysis.
|
| Growth protocol |
EndoC-βH2 cells (Scharfmann et al, 2014, JCI)) were cultured in low-glucose (5.6 mM) Dulbecco’s modified Eagle’s medium (DMEM; Sigma-Aldrich) containing L-glutamine and sodium pyruvate, supplemented with 2% BSA fraction V (Roche Diagnostics), 50 µM 2-mercaptoethanol, 10mM nicotinamide (Calbiochem), 5.5 µg/ml transferrin (Sigma-Aldrich), 6.7 ng/ml selenite (Sigma-Aldrich), 100 U/ml penicillin and 100 µg/ml streptomycin. Cells were seeded at a density of 5x104 cells/cm2 on Matrigel (1%) / fibronectin (2µg/ml) (Sigma-Aldrich) coated plates and cultured at 37°C and 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from EndoC-βH2 cells using RNeasy Micro kit (Qiagen).
|
| Label |
Cy3
|
| Label protocol |
One-Color Microarray-Based Gene Expression Analysis- Low Input Quick Amp Labeling (version 6.5, May 2010)
|
| |
|
| Hybridization protocol |
One-Color Microarray-Based Gene Expression Analysis- Low Input Quick Amp Labeling (version 6.5, May 2010). Complementary RNA (cRNA) samples were linearly amplified and labeled with cyanine3 starting from 50ng of total RNA. Following fragmentation, labeled cRNA were hybridized on Agilent “SurePrint G3 Human Gene Expression 8x60K Microarray” (Design ID 028004), for 17hrs at 65°C under 10rpm.
|
| Scan protocol |
AgilentG3_GX_1Color (scan resolution : 3 µm, Tiff : 20 bit). After washes, the slides were scanned using an Agilent G2565CA microarray Scanner System, at a 3μM resolution in a 20-bit scan mode, according to the “AgilentG3_GX_1Color” protocol.Raw.tif images were then extracted using Agilent “Feature Extraction, version 10.10.1.1” following “GE1_1010_Sep10” protocol.
|
| Description |
si RFX6 treated EndoC-βH2 cells
|
| Data processing |
The scanned images were analyzed with Feature Extraction sofware (Agilent) version 10.10.1.1 to obtain gprocessed signal data
|
| |
|
| Submission date |
Jul 03, 2014 |
| Last update date |
Dec 23, 2014 |
| Contact name |
Raphael Scharfmann |
| Organization name |
INSERM-Institut Cochin , Bâtiment Cassini
|
| Department |
U1016
|
| Street address |
123,boulevard de Port-Royal
|
| City |
Paris |
| State/province |
Paris |
| ZIP/Postal code |
75014 |
| Country |
France |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE59049 |
Transcriptome profile of human pancreatic beta cell line (EndoC-βH2 ) following knockdown of RFX6. |
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