|
| Status |
Public on May 01, 2020 |
| Title |
Dph5IR_3 |
| Sample type |
RNA |
| |
|
| Source name |
adult male whole gut expressing transgenes under the control of esg[ts]-Gal4, 2days at 29 degree
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: esg[ts]-Gal4>GFP, Dph5IR
tissue: whole gut
Sex: male
|
| Treatment protocol |
They were further incubated for two days at 29℃ to inactivate Gal80ts and allow regulation of gene expression by Gal4/UAS system.
|
| Growth protocol |
Flies were kept on standard diet at 18℃ for 8-14 days .
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from 15 whole guts of adult males by using QIAzol Lysis Reagent (QIAGEN#79306), followed by purification using RNeasy Plus Micro Kit (QIAGEN#74034).
|
| Label |
Cy3
|
| Label protocol |
Cyanine-3 (Cy3) labeled cRNA was prepared from 200 ng RNA using the Low Input Quick Amp Labeling Kit, One-Color (Agilent) according to the manufacturer's instructions, followed by RNeasy Mini Kit (QIAGEN). Dye incorporation and cRNA yield were checked with the NanoDrop 2000c Spectrophotometer.
|
| |
|
| Hybridization protocol |
Cy3-labelled cRNA was fragmented at 60°C for 30 minutes in a reaction volume of 25 μl containing 1x Agilent fragmentation buffer and 2x Agilent GE blocking agent following the manufacturer's instructions. On completion of the fragmentation reaction, 25 μl of 2x Agilent GE hybridization buffer HI-RPM was added to the fragmentation mixture and hybridized to SurePrint G3 custom microarray 8x60K for Drosophila for 17 hours at 65°C in a rotating Agilent hybridization oven. After hybridization, microarrays were washed 1 minute at room temperature with GE Wash Buffer 1 (Agilent) and 1 minute with 37°C GE Wash buffer 2 (Agilent), then dried immediately.
|
| Scan protocol |
Slides were scanned immediately after washing on the SureScan Microarray Scanner using AgilentG3_GX_1Color_HighSensitivity.
|
| Data processing |
The scanned images were analyzed with Feature Extraction Software 10.7.3.1 (Agilent) using default parameters (protocol GE1_107_Sep09 and Grid: 040871_D_F_20120511) to obtain background subtracted and spatially detrended Processed Signal intensities. Features flagged in Feature Extraction as Feature Non-uniform outliers were excluded. If the probes scored low values in Evaluation Metrics, the probes were removed (filtered).GeneSpring (Agilent), a microarray analysis software was used for normalizing the data.
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| |
|
| Submission date |
Nov 29, 2019 |
| Last update date |
May 02, 2020 |
| Contact name |
Miura Masayuki |
| Organization name |
The University of Tokyo
|
| Department |
Graduate School of Pharmaceutical Sciences
|
| Lab |
Genetics Lab
|
| Street address |
7-3-1 Hongo
|
| City |
Bunkyo-ku, Tokyo |
| ZIP/Postal code |
113-0033 |
| Country |
Japan |
| |
|
| Platform ID |
GPL17277 |
| Series (1) |
| GSE141201 |
Diphthamide modification of eEF2 is required for gut tumor-like hyperplasia induced by oncogenic Ras |
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