|
| Status |
Public on Apr 10, 2020 |
| Title |
eL3_EAD_NR_ey>Delta,ATF4RNAi_rep2 |
| Sample type |
RNA |
| |
|
| Source name |
Early L3 (150h AEL) larval eye-antenna disc.
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: ey-Gal4, UAS-Delta/+;ATF4-RNAi (BL-25985)/+
|
| Treatment protocol |
Larvae were dissected under a dissecting microscope, eye-antenna discs manually isolated and transferred to a tube with PBS and 0.001% Tween-20 on ice. When 20 to 30 eye-antenna discs (depending on size) were collected, supernatant was carefully removed and presence of discs in tube confirmed under the microscope. 1ml TRIzol was added and continously vortexed to allow fast and efficient lysis. Samples were then frozen at -80°C.
|
| Growth protocol |
Fly crosses were set up in small cages and females allowed to lay eggs on petri dishes with NR food (protein restricted, 20g fresh yeast per litre) for 2 to 3 hours. Larvae were then grown in the dishes until they reached the appropriate early L3 stage (150h AEL).
|
| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA extraction, samples were thawed and centrifuged at 12000rpm for 20' at 4°C. 1ml supernatant was transferred to a new tube containing 1ml 100% Ethanol. The mixture was then run stepwise through a ZymoResearch Direct-zol MicroPrep column, following the manufacturers protocol, including on-column DNAseI digest. Total RNA was eluted in 8µl pure water.
|
| Label |
biotin
|
| Label protocol |
50ng total RNA was used for in vitro amplification using Affymetrix GeneChip® 3’ IVT PLUS Reagent Kit according to the manufacturers specifications.
|
| |
|
| Hybridization protocol |
Samples were hybridized for 16 hours at 45°C (at 60rpm) on Drosophila Genome 2.0 Arrays. Arrays were washed and stained in the Affymetrix Fluidics Station 450 (Affymetrix GeneChip® Hybridization, Wash, and Stain Kit, catalogue number 900720).
|
| Scan protocol |
Arrays were scanned with a GeneChip Scanner 3000 7G.
|
| Data processing |
CEL files were interpreted with Affymetrix Expression Console (Build 1.4.1.46) using RMA normalisation (default settings). CHP files were used for gene expression analysis with Affymetrix Transcriptome Analysis Console (Version 3.0.0.466).
|
| |
|
| Submission date |
Apr 09, 2020 |
| Last update date |
Apr 10, 2020 |
| Contact name |
Sebastian Sorge |
| E-mail(s) |
[email protected]
|
| Organization name |
The Francis Crick Institute
|
| Street address |
1 Midland Road
|
| City |
London |
| ZIP/Postal code |
NW1 1AT |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE148414 |
Eye-antenna early L3 disc expression profiling in combinations of COX7a-LoF, ATF4-LoF and Notch-GoF |
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