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| Status |
Public on Jun 01, 2023 |
| Title |
Transcriptomic analysis of thoraces after overexpression of an active allele of REPTOR in muscle |
| Organism |
Drosophila melanogaster |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Purpose: identify global changes in gene expression in thorax tissues caused by an increase in activity of REPTOR in muscles. Note that the thorax of adult flies is composed mainly by muscle tissue but fat body is also present. Changes in gene expression do not exclusively reflect the muscle transcriptome
Methods: To extract total RNAs for RNA-Seq experiment, we used 5-6 thoraces dissected out from both tub-Gal80ts/+ ; dMef2-GAL4/+ (Con) and tub-Gal80ts/UAS-REPTOR[ACT] ; dMef2-GAL4/+ (REPTOR), making sure the gut of these flies was completely removed. Crosses were kept at 18°C to avoid expression of REPTOR during development. Adult males were collected every 24-48 hours and incubated 3-4 days at 18°C before being shifted to 29°C. Flies were then incubated for 4 days at 29°C. After assessing RNA quality with Agilent Bioanalyzer, mRNAs were enriched by poly-A pull-down. Then, sequencing libraries constructed with Illumina TruSeq RNA prep kit were sequenced using. We multiplexed samples in each lane, which yields targeted number of single-end 75 bp reads for each sample, as a fraction of 180 million reads for the whole lane. Sequence reads were mapped back to the Drosophila genome (flybase genome annotation version r6.30) using STAR. With the uniquely mapped reads, we quantified gene expression levels using Cufflinks (FPKM values). Next, differentially expressed genes between experimental and control data were analyzed with DESeq2.
Results: Gene list enrichment analysis of the downregulated thoracic transcriptome by REPTOR overexpression revealed a striking enrichment of multiple metabolic processes impinging on carbohydrate metabolism, mitochondria, glycolysis and oxidative metabolism. Also, Thor, a well-characterized target of REPTOR was upregulated and it was validated with qPCR.
Conclusions: Our study indicates that REPTOR is a strong regulator of muscle metabolism in adult flies.
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| Overall design |
Thoraces mRNA profiles of both tub-Gal80ts/+ ; dMef2-GAL4/+ (Con) and tub-Gal80ts/UAS-REPTOR[ACT] ; dMef2-GAL4/+ (REPTOR) flies incubated for 4 days at 29°C were generated by deep sequencing, in replicate, using Illumina HiSeq2000.
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| Web link |
https://pubmed.ncbi.nlm.nih.gov/37582831/
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| Contributor(s) |
Saavedra P, Norbert P |
| Citation(s) |
37582831 |
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| Submission date |
Nov 19, 2021 |
| Last update date |
Oct 26, 2023 |
| Contact name |
Yanhui Hu |
| E-mail(s) |
[email protected]
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| Phone |
617-432-6546
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| Organization name |
Harvard Medical School
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| Department |
Genetics
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| Lab |
Norbert Perrimon
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| Street address |
77 Avenue Louis Pasteur
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02115 |
| Country |
USA |
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| Platforms (1) |
| GPL19132 |
Illumina NextSeq 500 (Drosophila melanogaster) |
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| Samples (8)
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| Relations |
| BioProject |
PRJNA781995 |
| SRA |
SRP346955 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE189218_RAW.tar |
1.1 Mb |
(http)(custom) |
TAR (of TXT) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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