GSE107529
Next generation sequence analysis for transcriptome of Drosophila melanogaster third instar larval eye discs expressing activated Ras either alone or along with changes in hsromega  lncRNA levels
Purpose: While studying possible interaction between ras and hsr? genes we found that down- or up- regulation hsr? RNA levels results in further enhancement in activity hyperactive Ras protein in eye discs of Drosophila melanogaster. In order to find out reason underlying this phenomena we checked difference in gene expression between eye discs expressing activated Ras along with alterations in hsr? RNA levels and those expressing activated Ras alone. Method: Eye disc RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsr?RNAi, sev-GAL4>EP3037, sev-GAL4>UAS-RasV12, sev-GAL4>UAS-hsr?RNAi UAS-RasV12 and sev-GAL4>EP3037 UAS-RasV12 were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform using 50bp pair-end reads, 12 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 activated Ras in eye discs resulted in differential expression of many genes, with 374 genes being down-regulated and 138 up-regulated, when compared with those in sev-GAL4>UAS-GFP eye discs. Compared to only Ras expressing eye discs in case of down regulation of hsr? RNA in activated Ras background 148 gene were up-regulated while 434 were down-regulated. In eye discs expressing activated Ras alongwith over expression of hsr? RNA, 192 genes were up-regulated and 470 down-regulated compared to only activated Ras expressing eye discs. Some of these (11 genes) were validated using qRT-PCR. Conclusion: In activated Ras expressing eye discs, besides the expected increase in levels of transcripts of genes involved in cell growth, proliferation and differentiation, transcripts of many genes involved in RNA biosynthesis, metabolism and processing were up-regulated when compared with sev-GAL4>UAS-GFP eye discs. Levels of transcripts of none of the known members of Ras/Raf/MAPK signaling pathway were found to be significantly affected by changes in hsr? transcript levels in activated Ras expression background. Interestingly, several sn/snoRNAs along with a scaRNAs and some members of the RNA processing machinery were differentially expressed in activated Ras background and were further affected when the hsr? RNA levels were down-or up-regulated. In addition, while down-regulation of hsr? activity resulted in up-regulation of a few positive modulators of Ras signaling pathway, up-regulation of these transcripts caused down-regulation of the negative regulators of Ras/Raf/MAPK pathway, and thus resulting in increase in Ras activity in either cases. Overall design: Eye discs mRNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-RasV12, sev-GAL4>UAS-hsr?RNAi UAS-RasV12 and sev-GAL4>EP3037 UAS-RasV12 third instar larvae  were generated by sequencing, in triplicate, using Illumina Hiseq2500 platform.