|
| Status |
Public on Oct 01, 2017 |
| Title |
GIC2, control shRNA, rep 2 |
| Sample type |
RNA |
| |
|
| Source name |
proneural glioma stem-like cell_shRNA control
|
| Organism |
Homo sapiens |
| Characteristics |
tumor type: Glioblastoma
cell type: Patient-derived CD44low glioma stem-like initiating cell (GICs)
genotype/variation: control
|
| Treatment protocol |
Control- and GPR56 knockdown-GICs were obtained by infection with lentiviruses expressing either a non-mammalian shRNA control or a shRNA targeting GPR56, followed by puromycin selection.
|
| Growth protocol |
Patient-derived GICs were cultured under adherent conditions on laminin in serum-free neural stem cell medium (in the presence of bFGF and EGF) at 37°C, 5% CO2 and 5% oxygen.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen). RNA was quantified using a NanoDrop-1000 spectrophotometer and RNA quality control was monitored in a 2100 Bioanalyzer (Agilent Technologies). In all cases, the RNA integrity number was >7.
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.2 ug RNA using the One-Color Low Input Quick Amp Labeling kit (Agilent Technologies) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop ND-2000 Spectrophotometer.
|
| |
|
| Hybridization protocol |
1.5 μg of Cy3-labelled cRNA (specific activity >10.0 pmol Cy3/μg cRNA) was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 25x Agilent fragmentation buffer and 10x Agilent blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 μl of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent SurePrint G3 Human GE 8x60K Microarray (Agilent-039494) for 17 hours at 65°C in a rotating 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 DNA Microarray Scanner (G2565C) using one color (Cy3) scan setting for expression array slides (Scan Area 61x21.6 mm, Scan resolution 3um, Dye channel is set to Green and Green PMT is set to 100%).
|
| Description |
GIC-CTRL_04
gene expession of control glioma initiating cells in serum-free neural stem cell medium
|
| 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. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. Data normalization was carried out using GeneSpring GX version 11.5 (Agilent Technologies).
|
| |
|
| Submission date |
Feb 10, 2017 |
| Last update date |
Oct 01, 2017 |
| Contact name |
Nuria de la Iglesia |
| E-mail(s) |
[email protected]
|
| Organization name |
IDIBAPS
|
| Street address |
Rossello 153
|
| City |
Barcelona |
| ZIP/Postal code |
08036 |
| Country |
Spain |
| |
|
| Platform ID |
GPL16699 |
| Series (1) |
| GSE94765 |
GPR56/ADGRG1 inhibits Mesenchymal Differentiation and Radioresistance in Glioblastoma |
|
