GSE120077
Next generation sequence analysis for transcriptome of Drosophila melanogaster wandering third instar larva in wt and lncRNA hsromega mutant
Purpose: To compare transcriptomic changes between 3rd instar wild type larvae and those with highly down regulated hsr? lncRNAs levels Method: Whole third instar larval total RNA profiles of +/+ and +/+;hsromega[66] homozygous were generated by sequencing, in duplicate, on Illumina Hiseq2500 platform using 100bp pair-end reads, which resulted in a sequencing depth of ~20 million reads. The resulting 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. Cuffdiff 2.1.1 was used (Trapnell et al., 2012) to ascertain differential expression of gene transcripts between different samples, . Result: Using an optimized data analysis workflow, we mapped about 20 million sequence reads per sample to the Drosophila genome (dm6) and identified  21,529 transcripts with TopHat workflow in each genotype studied, with 20,956 having baseline expression. 134 genes were up regulated and 74 downregulated in +/+;hsromega[66] compared to +/+ whereas 201  expressed only in the wild type sample and 164 only in hsromega[66] mutant Conclusion: Analysis of RNA-seq data revealed that a large proportion of the transcripts affected in larvae with loss of hsromega lncRNA transcripts belonged to innate immune response pathway, mainly those in defense against Gram positive bacteria.  Many of these genes are part of proteolysis pathway having endopeptidase activity with predominence of serine-threonine endopeptidases. Kegg pathway analysis revealed that metabolism, especially that of Drugs, was indicated to be affected. Genes showing differential expression belonged to ascorbate and aldarate metabolism,retinol metabolism, pentose and glucoronate interconversion, metabolism of xenobiotics by cytochrome P450 Overall design: total RNA profiles of wandering late third instar larvae of +/+ and +/+;hsromega[66]  were generated by sequencing, in duplicate, using Illumina Hiseq2500 platform.