|
| Status |
Public on Nov 04, 2016 |
| Title |
Control_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
GSC-11 cell line, non-targeting control siRNA
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: GSC-11
sirna: non-targeting control
|
| Treatment protocol |
To inhibit Sox2 expression, transient transfection were performed using si-Sox2 or si-scramble (Ambion) according to the manufacturer's instructions using Lipofectamine 2000 (Invitrogen). The cells were then cultured for 72 h after transfections and subjected to RNA extraction
|
| Growth protocol |
GSC11 cells were maintained in Dulbecco's modified Eagle's medium/nutrient mixture F12 supplemented with B27 (Invitrogen, Carlsbad, CA), epidermal growth factor, and basic fibroblast growth factor (20 ng/mL each; Sigma-Aldrich, St Louis, MO.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total cellular RNA was isolated from the cultured cells using Trizol reagent (Ambion) according to the manufacturers' protocols
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 0.5 ug RNA using the One-Color Low RNA Input Linear Amplification PLUS kit (Agilent) according to the manufacturer's instructions, followed by RNAeasy column purification (QIAGEN, Valencia, CA).
|
| |
|
| 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 a reaction volume of 250 ml containing 1x Agilent fragmentation buffer and 2x Agilent blocking agent following the manufacturers instructions. On completion of the fragmentation reaction, 250 ml of 2x Agilent hybridization buffer was added to the fragmentation mixture and hybridized to Agilent Whole Human Genome Oligo Microarrays (G4112A) 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 by brief centrifugation.
|
| Scan protocol |
Agilent Technologies Scanner G2505C US82003505
|
| Data processing |
Data were normalized with Bioconductor software using quantiles
|
| |
|
| Submission date |
Mar 16, 2016 |
| Last update date |
Nov 04, 2016 |
| Contact name |
Marta Maria Alonso Roldán |
| E-mail(s) |
mmalonso@unav.es
|
| Organization name |
University Hospital of Navarra
|
| Department |
Dpt of Pediatrics
|
| Lab |
2.03
|
| Street address |
CIMA building , Pio XII 55
|
| City |
Pamplona |
| State/province |
Navarra |
| ZIP/Postal code |
31008 |
| Country |
Spain |
| |
|
| Platform ID |
GPL13607 |
| Series (1) |
| GSE79302 |
Analysis of Sox2-regulated transcriptome in glioma stem cells |
|
