GSE116476
Next-generation sequence analysis for transcriptome of Drosophila melanogaster third instar larval eye discs expressing sevenless-GAL4 directed down or up regulated levels of hsromega lncRNAs levels
Purpose: To compare transcriptomic changes in 3rd instar larval eye discs following sev-GAL4 driven down- or up- regulation of hsr? lncRNAs. Method: Eye disc total RNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsr?RNAi, 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 hsr? 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 hsr? RNA in normal as well as activated Ras expression background. A comparison of transcriptomes of sev-GAL4>hsr?RNAi 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-hsr?RNAi and sev-GAL4>EP3037 eye discs. Overall design: Eye discs mRNA profiles of wandering late third instar larvae of sev-GAL4>UAS-GFP, sev-GAL4>UAS-hsr?RNAi and sev-GAL4>EP3037 third instar larvae were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform.