|
| Status |
Public on Sep 02, 2014 |
| Title |
ESC control, biological rep1 |
| Sample type |
RNA |
| |
|
| Source name |
embryonic stem cells wild-type
|
| Organism |
Mus musculus |
| Characteristics |
tissue: embryonic stem cells
genotype: wild-type
|
| Growth protocol |
standard
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Invitrogen TRIzol, Qiagen RNeasy Mini kit
|
| Label |
biotin
|
| Label protocol |
RNA is converted into cDNA using a T7 promoter-tailed oligo-dT primer in the synthesis of the first cDNA strand; second strand cDNA synthesis is then carried out. The double-stranded cDNA is used as the template in an in vitro transcription (IVT) reaction catalyzed by T7 polymerase and containing biotinylated CTP and UTP in addition to the four unmodified ribonucleoside triphosphates.The biotinylated complementary RNA (cRNA) is purified from the IVT reaction mixture using magnetic particles. The cRNA is quantified spectrophotometrically and purity of the cRNA is also assessed by spectrophometric measurements. Those cRNA's that fall outside of an acceptable range will not routinely be carried forward in the analysis. Should this occur, investigators would be notified and asked how they would like to proceed.The purified cRNA is fragmented in order to facilitate the subsequent hybridization step.
|
| |
|
| Hybridization protocol |
The fragmented cRNA is added to a hybridization solution containing several biotinylated control oligonucleotides (for quality control), and hybridized to a microarray chip overnight at 45°C. The hybridization solution is then removed. The chips are transferred to a fluidics instrument that performs washes to remove cRNA that has not hybridized to its complementary oligonucleotide probe. The bound cRNA is then fluorescently labeled using phycoerythrin-conjugated streptavidin (SAPE); additional fluors are then added using biotinylated anti-streptavidin antibody and additional SAPE.
|
| Scan protocol |
not provided
|
| Data processing |
RMA normalized using the 'RMA' package in R
|
| |
|
| Submission date |
Jun 20, 2014 |
| Last update date |
Sep 04, 2014 |
| Contact name |
Tobias Otto |
| E-mail(s) |
tobias_mails@web.de
|
| Organization name |
Dana-Farber Cancer Institute
|
| Department |
Cancer Biology
|
| Lab |
Peter Sicinski
|
| Street address |
450 Brookline Avenue
|
| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02215 |
| Country |
USA |
| |
|
| Platform ID |
GPL18854 |
| Series (1) |
| GSE58712 |
Cyclin C is a haploinsufficient tumour suppressor |
|
