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| Status |
Public on Jul 13, 2010 |
| Title |
Mesodermal progenitors with inducible Notch1; OHT induced; CHX treated; replicate 2 |
| Sample type |
RNA |
| |
|
| Source name |
NERT mesodermal progenitors, w/ OHT 4h, w/ CHX 4 h
|
| Organism |
Mus musculus |
| Characteristics |
strain: EB5
cell type: mesodermal progenitor
nert: +
condition: Collagen+FCS
oht: +
chx: +
days differentiation: 4
|
| Treatment protocol |
For induction of Notch1 signaling, 1 µM tamoxifen (OHT) was added to the culture medium for 4 h. To block protein synthesis, 50 µg/ml cycloheximide (CHX) was added for 4 h in parallel to induction.
|
| Growth protocol |
Culture of undifferentiated ESC and the generation of mesodermal cells was performed as described (Schroeder et al. 2003, http://www.pnas.org/content/100/7/4018.long). Briefly, EB5 cells carring a tamoxifen inducible NERT construct or an empty control were kept under self-renewal conditions. For the culture of cells in conditions favoring ectodermal or mesodermal differentiation, ESC grown on gelatine were cultured for 4 h in GMEM, 10% Knockout Serum Replacement, 1% non essential amino acids, 1 mM sodium pyruvate and 100 µM 2-mercaptoethanol (ES to ectoderm), or in Knockout-DMEM, 10% Knockout Serum Replacement, 1% pre-tested FCS, 1% non essential amino acids and 100 µM 2-mercaptoethanol (ES to mesoderm). Mesodermal progenitors were generated by transferation of EB5 cells into mesodermal differentiation medium on Collagen IV coated dishes and kept for 4 d. Successful differentiation was monitored by generation of Flk1+ cells via FACS analysis.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using RNeasy Midi kit (Qiagen, Hilden, Germany) with DNAse digestion using the maufacturer's buffers and protocols. RNA quality was assessed by Agilent 2100 Bioanalyzer System.
|
| Label |
biotin
|
| Label protocol |
Probe preparation was with 5 microgram RNA using the Affymetrix One-Cycle Target Labeling Kit according to the manufacturer's instructions. Labeled RNA was fragmented by heating to 95°C for 35 minutes in 200 mM TRIS (pH 8.1), 500 mM KOAc, 150 mM MgOAc buffer.
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| |
|
| Hybridization protocol |
Affymetrix arrays were prehybridized with 200 microliter MES hybridization buffer for 10 minutes at 45°C with rotation (60 U/min) in an Affymetrix Hybridization Oven 640. After removing the prehybridization solution, 15 microgram of labeled cRNA in 200 microliter MES hybridization buffer were applied to the array leaving a small air bubble. Arrays were incubated overnight at 45°C with rotation (60U/min) in the hybridization chamber.
|
| Scan protocol |
Arrays were scanned using Affymetrix GeneChip Scanner 3000.
|
| Description |
Gene expression data from mesodermal progenitors
|
| Data processing |
The data were analyzed with dChip software (http://www.dchip.org/; Built date:Aug 21 2008) using dChip default analysis settings and were normalized to an array with median overall intensity ('Con_M+' = ‘Control Mesoderm +OHT’) using the Invariant Set Normalization method.
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| |
|
| Submission date |
Mar 17, 2009 |
| Last update date |
Jul 13, 2010 |
| Contact name |
Ralf Schwanbeck |
| E-mail(s) |
[email protected]
|
| Organization name |
University of Kiel
|
| Department |
Biochemistry
|
| Street address |
Otto-Hahn-Platz 9
|
| City |
Kiel |
| ZIP/Postal code |
24118 |
| Country |
Germany |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE15268 |
Cell-context dependent Notch target genes |
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