|
| Status |
Public on Apr 13, 2015 |
| Title |
Wounded/GFP+, replicate B |
| Sample type |
RNA |
| |
|
| Source name |
Wing discs, wounded, GFP+
|
| Organism |
Drosophila melanogaster |
| Characteristics |
strain/background: PucE69-Gal4 I; UAS-GFP (yw; UAS-GFP; pucE69-Gal4 I/TM6B)
tissue: wing imaginal disc cells
developmental stage: third instar larvae
treatment: wounded
gfp: positive (Gal4-UAS system, puc enhancer)
|
| Treatment protocol |
Third instar larvae imaginal disc dissections were performed in cultured medium and incisions of precise sizes were carefully executed with a pair of tungsten needles.This was followed by static culture in supplemented MM3 media for 16-18 hours. Before sorting cells by FACS, imaginal discs were dissociated by trypsinization. Trypsinization was done in 35 mm dishes (Nunclon) with 1 ml of Trypsin-EDTA (Sigma –T4174) 9 X and 5 ml of Hoechst 33342 (final dilution 0.1 μg/ml). Around 100 imaginal discs were shaked 2-3 hours (170 rpm) at room temperature. During the last 30 minutes, 5 ml of propidium iodide (1 mg/ml, Invitrogen) were added to detect dead cells. The degree of dissociation was monitored under the scope. The selection and sorting of GFP-positive and -negative cells were done by Flow Cytometry using a MoFlo equipment based on drop formation (DakoCytomation).
|
| Growth protocol |
Third instar larvae wing imaginal discs were dissected and incubated in a MM3 medium (Shields and Sang's) supplied with fly extract.The fly extract was prepared following the protocol suggested by the DGRC, Indiana University (Lin G, Zhang X, Ren J, Pang Z, Wang C, et al. (2013) Integrin signaling is required for maintenance and proliferation of intestinal stem cells in Drosophila. Dev Biol 377: 177-187). This modified media allows the culture of the wing imaginal discs for more than 24 hours.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted using the Qiagen RNeasy® Micro Kit. In order to obtain approximately 10 ng of RNA, we employed around 30,000 to 40,000 cells. The quality and quantity of the extracted RNA were analyzed using the RNA Pico Lab Chip with an Agilent Bioanalyzer (Agilent Technologies).
|
| Label |
biotin
|
| Label protocol |
Due to the small size of the sample, the RNA was indirectly labeled with two rounds of linear amplification.
|
| |
|
| Hybridization protocol |
The resulting cRNAs were hybridized to GeneChip Drosophila Genome 2.0 Arrays (Affymetrix, Santa Clara, CA).
|
| Scan protocol |
GeneChips were scanned.
|
| Description |
Strain PucE69-Gal4 I; UAS-GFP (yw; UAS-GFP; pucE69-Gal4 I/TM6B): Pastor-Pareja et al. (2004) Developmental Cell 7: 387-399.
Third instar larvae wing imaginal disc cells.
|
| Data processing |
Microarrays raw intensities were converted to gene expression estimates using the Robust Multichip Average procedure (RMA) on the FIESTA web server: Oliveros, J.C. (2007). FIESTA@BioinfoGP. An interactive server for analyzing DNA microarray experiments with replicates.
http://bioinfogp.cnb.csic.es/tools/FIESTA
|
| |
|
| Submission date |
Oct 30, 2014 |
| Last update date |
Apr 13, 2015 |
| Contact name |
Enrique Blanco |
| E-mail(s) |
[email protected]
|
| Phone |
+34 93 316 01 00
|
| Organization name |
Center for Genomic Regulation (CRG)
|
| Department |
Gene Regulation, Stem Cells and Cancer
|
| Lab |
Epigenetic Events in Cancer (L. Di Croce's lab)
|
| Street address |
Dr. Aiguader 88
|
| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE62863 |
Identification and Functional Analysis of Healing Regulators in Drosophila |
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