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| Status |
Public on Apr 10, 2015 |
| Title |
Cut, 12h-cultured, P3/4, biological rep3 |
| Sample type |
RNA |
| |
|
| Source name |
Prothorasic leg discs of 100h-old male Drosophila larvae
|
| Organism |
Drosophila melanogaster |
| Characteristics |
genotype: w/Y; AyGAL4, UAS-GFP/+; puc[E69-I]-GAL4/UAS-flp
gender: male
developmental stage: 100h after embryo laying
tissue: Prothoracic leg imaginal disc
treatment: Cut, 12h-cultured
region: posterior 3/4
|
| Treatment protocol |
To minimize the time variance among each sample preparation, disc fragmentation and transplantation were performed within one hour.
|
| Growth protocol |
To obtain a stage-synchronized cohorts of larvae, embryos were collected in one hour and incubated for exactly 100 hours at 25˚C. Cut or uncut discs were cultured in female adult fly abdomens for exactly 12 or 24 hours at 25˚C.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA of regenerating cells or control cells were extracted using PicoPureTM RNA Isolation Kit (ARCTRUS/Life Technologies).
|
| Label |
biotin
|
| Label protocol |
Biotinylated cRNA were prepared according to the standard Affymetrix protocols of GeneChip® Two-Cycle CDNA Synthesis kit and GeneChip® IVT labeling kit (Affymetrix).
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| |
|
| Hybridization protocol |
cRNA (10 µg) was hybridized for 16 hr at 45˚C on GeneChip® Drosophila Genome 2.0 Array. Following hybridization, the arrays were processed using a GeneChip Fluidics Station 400 (Affymetrix).
|
| Scan protocol |
Fluorescent images of arrays were captured using the GeneChip® Scanner 3000 7G (Affymetrix)
|
| Description |
Prothorasic leg discs of 100h-old male Drosophila larvae were cut to P3/4 fragments and 12h-cultured, and the GFP-positive region (regenerating cells) were isolated.
|
| Data processing |
Microarray data were preprocessed and analyzed using R and BioConductor. First, diagnostic plots were produced and used to exclude spatial artifacts. Then, probeset level intensities were background subtracted and quantile-quantile normalized using RMA. Hierarchical clustering analysis of expression values indicated the presence of batch effects. Data were therefore adjusted for this systematic error by performing a gene-wise one-way analysis of variance using PAMR (Prediction Analysis of Microarrays, for R) (Tibshirani R. et al., PNAS 99:6567-6572 (2002)). Differential expression analysis was carried out by computing moderated F-statistics using limma. Test p-values were adjusted for multiple comparisons using the Benjamini-Hochberg correction and genes with adjusted p < 0.001 were deemed significantly differentially expressed in at least a core comparison (C12A-UC12A, C24A-UC24A, C12P-UC12P, or C24P-UC24P). Series supplementary files 'anova_p10-3_4core-comparisons_YableS1.txt' and 'anova_p10-3_4culturing.txt' list expression values, defined as gene-wise one-way ANOVA residuals (after batch effect removal with PAMR).
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| |
|
| Submission date |
Feb 11, 2014 |
| Last update date |
Apr 10, 2015 |
| Contact name |
Tomonori Katsuyama |
| E-mail(s) |
[email protected]
|
| Organization name |
ETH Zurich
|
| Department |
D-BSSE
|
| Street address |
Mattenstrasse 26
|
| City |
Basel |
| ZIP/Postal code |
4055 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL1322 |
| Series (1) |
| GSE54868 |
JAK/STAT coordinates cell proliferation during disc regeneration with Dilp8-mediated developmental delay in Drosophila melanogaster |
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