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Dmyc overexpression 7h and 14h

ABSTRACT: Fly strains: All transgenes are P[+] in w strains. w+;+;Act 5c > CD2 > GAL4 UAS-GFP (Neufeld et al. 1998 ); y w hs-FLP122; +; UAS-dMyc (Zaffran et al. 1998 ). y w hs-FLP122; +; +. Adult flies and larvae were raised in regular fly food consisting of cornmeal and molasses at 25°C. Larvae overexpressing either UAS-regulated dMyc;GFP or GFP alone transgenes were generated using the Flp/Gal4 method (Struhl and Basler 1993 ; Pignoni and Zipursky 1997 ; Neufeld et al. 1998 ). Larvae were staged from hatching and raised at a density of 50 per vial at 25°C. Third instar larvae (110 h after egg deposition, AED) were heat shocked at 37°C for 2 h, and larvae were collected 7 h after heat shock (~120 h AED). Total RNA was isolated using TRIzol reagent (Invitrogen) as described by manufacturer followed by RNeasy (Qiagen) clear up. cRNA targets were generated using a standard amino-allyl labeling protocol, where 30 µg each of "experimental" (dMyc;GFP: hs-FLP122; Act-GAL4, UAS-GFP; UAS-dMyc) and "reference" (GFP only :ywhs-FLP122; Act-GAL4, UAS-GFP; +) total RNAs were coupled to either Cy3 or Cy5 fluorophores. Paired labeled targets were processed on microarrays using protocols described elsewhere (Fazzio et al. 2001 ). Posthybridized arrays were scanned using a GenePix 4000 scanner (Axon Instruments). Data were generated from five independent replicates (two with one dye orientation and three with the reversed dye orientation) at 7h and 14h Keywords: repeat sample

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE2073 | GEO | 2004/12/16

SECONDARY ACCESSION(S): PRJNA91275

REPOSITORIES: GEO

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Project description:Purpose: To compare transcriptomic changes in 3rd instar larval eye discs following sev-GAL4 driven down- or up- regulation of hsrω lncRNAs. Method: Eye disc total RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsrωRNAi, sev-GAL4>EP3037 were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 6 samples per lane and each sample run across 2 lanes. This resulted in a sequencing depth of ~20 million reads. The resulting sequencing FastQ files were mapped to the Drosophila genome (dm6) using Tophat with Bowtie. The aligned SAM/BAM file for each was processed for guided transcript assembly using Cufflink 2.1.1 and gene counts were determined. Mapped reads were assembled using Cufflinks. Transcripts from all samples were subjected to Cuffmerge to get final transcriptome assembly across samples. In order to ascertain differential expression of gene transcripts between different samples, Cuffdiff 2.1.1 was used (Trapnell et al., 2012). Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified 15,905 transcripts with TopHat workflow in each genotypes studied. The sev-GAL4 driven expression of RNAi for hsromega gene transcripts in eye discs resulted in differential expression of many genes, with 409 genes being down-regulated and 100 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to control eye discs in case of up regulation of hsrω RNA, 66 gene were up-regulated while 635 were down-regulated. Some of these (11 genes) were validated using qRT-PCR. Conclusion: Analysis of RNA-seq data revealed that a large proportion of transcripts were indeed similarly affected by down- or up-regulation of hsrω RNA in normal as well as activated Ras expression background. A comparison of transcriptomes of sev-GAL4>hsrωRNAi and sev-GAL4>EP3037 eye discs revealed that in each case the number of genes down regulated was more than those up-regulated. Interestingly, while 319 genes were commonly down regulated and 15 were commonly up-regulated in the two genotypes, only 2 genes showed opposing trends between sev-GAL4>UAS-hsrωRNAi and sev-GAL4>EP3037 eye discs.

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Dmyc overexpression 7h and 14h

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