|
| Status |
Public on Dec 03, 2013 |
| Title |
control_RIP_Chip3 |
| Sample type |
RNA |
| |
|
| Source name |
Oregon-R wild type 0-3hr embryo control RIP RNA
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain: Oregon-R
genotype/variation: wild type
developmental stage: 0-3 hr embryo
temperature: 25 °C
rip antibody: control
|
| Growth protocol |
Oregon-R wild-type fly stocks were used. All stocks were maintained at 25 °C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Embryos collected at 0-3 hours post-egglaying were dechorionated with 50% bleach and homogenized in a minimal volume of RIP lysis buffer (150 mM KCl, 20 mM HEPES pH 7.4, 1 mM MgCl2, 1 mM DTT, 1x protease inhibitor cocktail (Bioshop)). Extracts were centrifuged for 10min at 4°C, and the supernatant was supplemented with 9 M urea to a final concentration of 2 M. Protein A beads were pre-incubated with either guinea pig anti-Smaug antibody or normal guinea pig serum followed by four washes with RIP lysis buffer supplemented with urea. These beads were then incubated with embryo extract for 2 h at 4°C followed by four washed with RIP lysis buffer supplemented with urea and RNA was extracted from the beads using the Trizol reagent (Invitrogen).
|
| Label |
Cy5
|
| Label protocol |
The cDNAs were prepared following the indirect labeling protocal provided by the Canadian Drosophila Microarray Centre using anchored-oligo-dT primers. Cy3 or Cy5-tagged random nonamers were then used to labeled the cDNA.
|
| |
|
| Hybridization protocol |
Labeled cDNA was used to probe Drosophila 4 x 72K NimbleGen arrays: Expression design for Drosophila melanogaster (Release 4.3) (GEO platform number: GPL13782).
|
| Scan protocol |
The arrays were scanned using GenePix4000B microarray scanner system (Molecular Devices, Inc., Sunnyvale, CA, USA)
|
| Description |
Sample name: control_03
Drosophila_melanogaster_0-3 hr_embryo_control_RIP_Chip3
control RIP-Chip biological replicate 2
|
| Data processing |
The raw values for the arrays were quantified using Nimblescan (Roche), following the protocol described in the NimbleGen Array User's Guide (Gene Expression Arrays, version 5.0). Arrays were normalized by Robust Multi-array Average (RMA) provided in the Nimblescan software.
|
| |
|
| Submission date |
Aug 16, 2013 |
| Last update date |
Dec 03, 2013 |
| Contact name |
Howard D Lipshitz |
| E-mail(s) |
howard.lipshitz@utoronto.ca
|
| Organization name |
University of Toronto
|
| Department |
Molecular Genetics
|
| Street address |
1 King's College Circle
|
| City |
Toronto |
| State/province |
ON |
| ZIP/Postal code |
M5S1A8 |
| Country |
Canada |
| |
|
| Platform ID |
GPL13782 |
| Series (1) |
| GSE49943 |
Identification of Smaug target mRNAs in the early Drosophila embryo using RIP-Chip |
|
