|
| Status |
Public on Jul 08, 2016 |
| Title |
Sox2+ cells, biological rep4 |
| Sample type |
RNA |
| |
|
| Source name |
Sox2+ Patched1+/- medulloblastoma cells
|
| Organism |
Mus musculus |
| Characteristics |
genotype/variation: Patched1+/- CD1; Sox2eGFP B6 parents
age: >70 days
cell type: medulloblastoma cells
cell subpopulation: Sox2-eGFP+ cells
|
| Treatment protocol |
Upon exhibiting signs of a brain tumour, animals were sacrificed using CO2 asphixiation and tumours microdissected in PBS. Tumours were dissociated to single cells using gentle pipetting followed by straining through a 70um then 40um cell strainer. Cell suspensions were depleted of CD45 and Ter119+ cells and Sox2 GFP+ and Sox2 GFP- cells were isolated by FACS on a Beckman Coulter MoFlo 9 colour cell sorter.
|
| Growth protocol |
Mice were housed according to CCAC standards at Sickkids Laboratory for Animal Services. Patched1+/-; Sox2eGFP mice were administered 3Gy ionizing radiation at birth to induce medulloblastoma formation.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Sorted cells were resuspended in 333uL Trizol and frozen at -80C. On the day of sample preparation, 3 samples, each representing one tumour, per biological replicate (GFP+ or GFP-) were pooled and RNA extracted using the ZymoReagent Easy-Zol column purification kit
|
| Label |
biotin
|
| Label protocol |
cDNA were prepared from 0.1ug RNA with the Ambion WT Gene Expression kit.
|
| |
|
| Hybridization protocol |
Following fragmentation, 5.5 ug of cDNA were hybridized to the Affymetrix Mouse Gene 2.0 ST (GPL16570) array using the FS450_0002 protocol
|
| Scan protocol |
GeneChips were scanned using the Affymetrix GeneChip Scanner 3000.
|
| Description |
Sox2_Positive_4
|
| Data processing |
CHP files were generated by Expression Console (v.1.2.0.20) using Affymetrix default analysis settings and Robust Multi-array average (RMA) as normalization method. Partec was used for to analyze CEL files using RMA normalization for prinicple component analysis, discovery of differentially expressed genes by one-way ANOVA and hierarchical clustering.
|
| |
|
| Submission date |
Jul 10, 2013 |
| Last update date |
Jul 08, 2016 |
| Contact name |
John E Dick |
| Organization name |
University of Toronto
|
| Department |
Molecular Genetics
|
| Lab |
John Dick Lab
|
| Street address |
University Health Network, Princess Margaret Cancer Research Tower, E Tower, 8th floor, 8-301
|
| City |
Toronto |
| State/province |
Ontario |
| ZIP/Postal code |
M5G1L7 |
| Country |
Canada |
| |
|
| Platform ID |
GPL16570 |
| Series (1) |
| GSE48766 |
Gene expression profiling of Sox2+ Patched1+/- medulloblastoma cells |
|
