|
| Status |
Public on Dec 31, 2012 |
| Title |
20060401_B.1.RhoV14.over |
| Sample type |
RNA |
| |
|
| Source name |
Drosophila S2R+ cells transfected with act-GAL4, UAS-GFP and plasmid encoding RhoV14 (constitutively active)
|
| Organism |
Drosophila melanogaster |
| Characteristics |
cell line: S2R+
cell line derivation: late stage embryos
|
| Treatment protocol |
RNAi experiments were performed by transfecting S2R+ cells with specific dsRNAs in combination with actin-GAL4 and UAS-GFP plasmids. Alternatively, cells were transfected with actin-GAL4 and UAS-GFP plasmids in addition to plasmids encoding activated forms of Rho-family GTPases, RhoGEFs, or RhoGAPs. All transfections were performed using Effectene Reagent (Qiagen). Following plasmid/RNAi transfection, cells were incubated for 5 days at room temperature. S2R+ cells were transfected in batches of 4-6 over the course of several months.
|
| Growth protocol |
Drosophila S2R+ cells were cultured in Schneider’s media (Sigma), supplemented with 10% Fetal Bovine Serum (SAFC Biosciences), and Penicillin-Streptomycin (Gibco).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated using TRIzol Reagent (Invitrogen) as directed by the manufacturer. Double-stranded cDNA incorporating a T7 RNA Polymerase promoter site was prepared from total RNA using the Roche Applied Sciences Microarray cDNA Synthesis Kit and purified using the Qiagen QIAquick purification kit.
|
| Label |
biotin
|
| Label protocol |
The Ambion MegaScript Kit was used to produce biotin-labeled antisense RNA (aRNA) by combining Biotin-11-UTP (PerkinElmer) and Biotin-CTP (PerkinElmer) with unlabelled UTP and CTP in a molar ratio of 1:3. Biotin-labeled aRNA was purified using the Qiagen RNeasy kit.
|
| |
|
| Hybridization protocol |
The aRNA mixtures were fragmented in a solution of 40 mM Tris-Acetate pH8.1, 100 mM KOAc, 30 mM MgOAc) at 95°C for 20 minutes, then placed on ice. Hybridization to Combimatrix CustomArrayä microarrays was performed as described by the manufacturer at 45°C for 18 hours under gentle rotation in a Fisher Biotech Hybridization Incubator under gentle rotation. Following hybridization and washing, arrays were blocked in 2XPBS, 0.1% Tween-20, 1% Acetylated BSA (Sigma) for 15 minutes at room temperature (RT), and subsequently incubated with Streptavidin-Alexa Fluor 647 (Molecular Probes) at final concentration of 1 μg/ml in the blocking solution for 30 minutes at RT while protected from light. Two subsequent washing steps were then performed at RT for 5 minutes using 2X PBS, 0.1% Tween-20 followed by 2 rounds of washing using 2X PBS with no detergent.
|
| Scan protocol |
Arrays were scanned using the Axon Genepix 4000B scanner.
|
| Description |
20060401_B_1
S2R+ cells have been repeatedly passaged in the Perrimon laboratory for several years.
|
| Data processing |
Median expression was extracted, spatial artifacts were removed by iterative surface-fit, features were averaged by probe sequence, and Loess-normalized with span=0.4.
|
| |
|
| Submission date |
Sep 28, 2009 |
| Last update date |
Dec 31, 2012 |
| Contact name |
Michael Baym |
| E-mail(s) |
baym@mit.edu
|
| Organization name |
MIT
|
| Department |
Mathematics
|
| Lab |
Berger
|
| Street address |
MIT 32-G572 / 32 Vassar St.
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
| |
|
| Platform ID |
GPL7159 |
| Series (1) |
| GSE18307 |
Systems Modeling of the Rho Signaling Network |
|
