|
| Status |
Public on Apr 18, 2015 |
| Title |
dissected_bD18ED_1 |
| Sample type |
RNA |
| |
|
| Source name |
D18 IA embryos, bD18ED, dissected
|
| Organism |
Bos taurus |
| Characteristics |
tissue: D18 IA embryos
origin: dissected
cell type: bD18ED
|
| Biomaterial provider |
INRA/UMR Biologie du Developpement et Reproduction
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. IVT amplified RNA from each sample was synthesized with the MessageAmpTM aRNA Kit (Ambion) according to Degrelle et al., 2008 (RT on 1µg total RNA, IVT during 10h). aRNA was purified on Mini Quick Spin RNA columns (Roche Diagnostic).
|
| Label |
33P
|
| Label protocol |
aRNA was retro-transcribed and directly labelled with [alpha-33P]dATP as described (Degrelle et al., 2008). 500ng of aRNA was mixed with 500ng of random hexamers in a volume of 25µl. The mixture was incubated at 70°C for 10 min and chilled on ice. cDNA was synthesised by the addition of 5µl 10X PCR buffer, 5µl 25mM MgCl2, 5 µl 0,1 mM DTT, 2,5µl 10mM mix dGTP, dCTP and dTTP, 2,5µl water, 50 µCi [alpha-33P]dATP and 200U Superscript II (Invitrogen) at 42°C for 50min. The RNA template was removed by the addition of 1µl RNAse H- and incubation at 37°C for 20 min. Labelled targets were then purified on Sephadex columns (G-50).
|
| |
|
| Hybridization protocol |
Prehybridisation in ExpressHybTM Hybridization Solution (Clontech) at 68°C during 1h (14ml). Hybridisation using new ExpressHybTM Hybridization Solution (Clontech) at 68°C overnight (10ml). Arrays were washed four times in 2X SSC, 1% SDS and once in 0.1X SSC, 0.5% SDS at 68°C for 30 min each. They were then exposed to phosphor-screens (Amersham) for 5 days.
|
| Scan protocol |
Scanner: STORM 760 from Molecular Dynamics - Raw data set : Feature extraction with Imagene 5.5 software from BioDiscovery (Proteigene)
|
| Description |
Syntax of the sample titles: Origin_CellType_ReplicateNumber - Where the Origin is either cultured, microdissected (cells) or dissected (tissues); Where the Cell type is either bTC, bXEC, bXMC or bD18ED (bovine day-18 embryonic disc).
|
| Data processing |
Intensity = Signal Mean - Normalization = Signal mean were log2 transformed and normalized by the mean of all the intensities on the array.
|
| |
|
| Submission date |
Dec 04, 2013 |
| Last update date |
Apr 18, 2015 |
| Contact name |
Severine Aude Degrelle |
| E-mail(s) |
severine.degrelle@inserm.fr
|
| Organization name |
INSERM UMRS-1139
|
| Street address |
4 avenue de l'Observatoire
|
| City |
PARIS |
| ZIP/Postal code |
75006 |
| Country |
France |
| |
|
| Platform ID |
GPL7417 |
| Series (1) |
| GSE52967 |
Primary bovine extra-embryonic cultured cells: a new resource for the study of in vivo peri-implanting phenotypes and mesoderm formation |
|
