|
| Status |
Public on Sep 19, 2007 |
| Title |
EBs with doxy replica B |
| Sample type |
RNA |
| |
|
| Source name |
EBs day6 withdoxycycline, experiment_B
|
| Organism |
Mus musculus |
| Characteristics |
HoxB4-inducible ES cells
|
| Treatment protocol |
After 4 days in culture at 37ºC, Hoxb4 was induced by addition of doxycycline to 2 ug/ml, and further incubated for 48 hours. After 48 hours the celly were lysed in RNAzol and total RNA was purified.
|
| Growth protocol |
The Hoxb4i ES cell line (Kyba et al. 2002, Cell 109:29-37) was cultured in DMEM supplemented with 0.1 mM non-essential aminoacids, 2 mM glutamine, 15% FCS, 0,1 mM beta-mercaptoethanol and 103 U/ml of LIF. Embryoid bodies were made by trypsinizing ES cells and incubating them in IMDM supplemented with 15% FCS, 200 ug/ml transferrin, 4.5 mM monothioglycerol, 50 ug/ml ascorbic acid and 2 mM glutamine at a concentration of 3500 cells/ml in bacteriological plates. After 4 days in culture at 37ºC, Hoxb4 was induced by addition of doxycycline to 2 ug/ml, and further incubated for 48 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from EBs using RNAzol according to the manufacturer´s recommendations. RNA integrity was confirmed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay (Agilent Technologies).
|
| Label |
Biotin
|
| Label protocol |
RNA was processed for use on Affymetrix (Santa Clara, CA, USA) GeneChip Mouse Genome 430 2.0 Arrays, according to the manufacturer’s One-Cycle Target Labeling Assay. Briefly, 4.3 µg of total RNA containing spiked in Poly-A RNA controls (GeneChip Expression GeneChip Eukaryotic Poly-A RNA Control Kit; Affymetrix) was used in a reverse transcription reaction (One-Cycle DNA synthesis kit; Affymetrix) to generate first-strand cDNA. After second-strand synthesis, double-stranded cDNA was used in an in vitro transcription (IVT) reaction to generate biotinylated cRNA (GeneChip Expression 3’-Amplification Reagents for IVT-Labeling; Affymetrix). Size distribution of the cRNA and fragmented cRNA, respectively, was assessed using an Agilent 2100 Bioanalyzer with a RNA 6000 Nano Assay.
|
| |
|
| Hybridization protocol |
15 µg of fragmented cRNA was used in a 300-µl hybridization containing added hybridization controls. 200 µl of mixture was hybridized on arrays for 16 h at 45°C. Standard post hybridization wash and double-stain protocols (EukGE-WS2v5) were used on an Affymetrix GeneChip Fluidics Station 400.
|
| Scan protocol |
Arrays were scanned on an Affymetrix GeneChip scanner 3000 for gene expression profiling as set forth by the manufacturer’s recommendations.
|
| Description |
Doxycycline-induced, day 6 Embryoid bodies (EBs) made from iHoxB4 ES cells, experiment B
|
| Data processing |
Scanned arrays were analyzed with Affymetrix MAS 5.0 software using default settings from Affymetrix. Normalized by global scaling, trimmed mean target intensity set to 500.
|
| |
|
| Submission date |
Sep 14, 2007 |
| Last update date |
Aug 28, 2018 |
| Contact name |
Moises Mallo |
| E-mail(s) |
[email protected]
|
| Organization name |
Instituto Gulbenkian de Ciencia
|
| Lab |
Patterning and Morphogenesis
|
| Street address |
Rua da Quinta Grande 6
|
| City |
Oeiras |
| ZIP/Postal code |
2780-156 |
| Country |
Portugal |
| |
|
| Platform ID |
GPL1261 |
| Series (1) |
| GSE9044 |
HOXB4 target genes in ES cell-derived embryoid bodies (EBs) |
|
| Relations |
| Reanalyzed by |
GSE119085 |
