|
| Status |
Public on Nov 01, 2014 |
| Title |
rNCSC-2-rep1 |
| Sample type |
RNA |
| |
|
| Source name |
Neurosphere cultures from embryonic DRG
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
tissue: embryonic day E12.5 DRG
|
| Treatment protocol |
rNCSCs and pNCSCs were cultured in serum-free proliferation medium: DMEM/F12, bFGF (20 or 40ng/ml), EGF (20 ng/ml), heparin (0.5 U/m)l, N2 (1%), B27 (1% or 3% ), Glutamine (1%) and Penicillin/Streptomycin (1%).
|
| Growth protocol |
DRG were isolated from C57BL/6J mice at embryonic day 12.5; palatal tissue was dissected from 3 to 5 C57BL/6J mice of the age of 6 to 8 weeks. Tissue was dissociated enzymatically, followed by mechanical trituration. Dissociated cells were cultured in proliferation medium. After 5–7 d in vitro, primary neurospheres were collected, incubated in accutase for 30 min, dissociated into single cells and plated for secondary sphere formation. For microarray analysis, passage 3 neurospheres were used.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted and purified by using a combination of TRIzol reagent (Life Technologies) and RNeasy MinElute Spin Columns (Quiagen) according to the manufacturers’ instructions.
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocol from 500 ng total RNA (Expression Analysis Technical Manual, 2001, Affymetrix).
|
| |
|
| Hybridization protocol |
Following fragmentation, 20 µg of cRNA were hybridized on GeneChip Mouse 430_2 arrays. GeneChips were washed and stained in the Affymetrix Fluidics Station 400.
|
| Scan protocol |
Fluorescence signals were recorded by an Affymetrix scanner 3000.
|
| Description |
DRG-derived NCSCs are reprogrammed in neurosphere cultures to rNCSCs that can give rise to CNS progeny including Olig2+ cells with oligodendrocyte morphology and markers O4, CNPase and Sox10.
|
| Data processing |
All further analysis was performed in R (http://www.r-project.org) using packages from the Bioconductor project. Functions from the affyPLM package were used for quality assessments of the microarrays and only good quality arrays were further analysed. GeneChip raw expression values were normalized and summarized using the GC-RMA method.
|
| |
|
| Submission date |
Apr 23, 2014 |
| Last update date |
Nov 01, 2014 |
| Contact name |
Galina Apostolova |
| Organization name |
Medical University Innsbruck
|
| Department |
Institute for Neuroscience
|
| Street address |
Innrain 66
|
| City |
Innsbruck |
| ZIP/Postal code |
6020 |
| Country |
Austria |
| |
|
| Platform ID |
GPL1261 |
| Series (2) |
| GSE57001 |
Generation of CNS neural stem cells and PNS derivatives from neural crest derived peripheral stem cells [Dataset 2] |
| GSE57003 |
Generation of CNS neural stem cells and PNS derivatives from neural crest derived peripheral stem cells |
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