RNA-seq identifies genes that are regulated by SAGA deubiquitinase activity in glial nuclei of the Drosophila central nervous system during larval development

Purpose: Next-generation sequencing (NGS) was used to elucidate genes in Drosophila visual system glia that are sensitive to impairment of the deubiquitinase activity of the Spt-Ada-Gcn5 Acetyltransferase (SAGA) complex. Methods: The nuclear envelope of glial cells was genetically labled with a KASH-GFP fusion protein using the Gal4/UAS binary expression system and repo-Gal4 as driver in wild-type, nonstop and sgf11 third instar larvae. GFP-labeled glial nuclei were released by homogenization and then isolated from the central nervous system/eye antennal disc complex of Drosophila melanogaster third instar larvae from each genotype, and mRNA was extracted. For RNA-seq, dsDNA was generated using the Ovation RNA-Seq System V2 (Nugen technologies), DNA libraries were generated using standard methods (Illumina), and paired-end 100-base fragements were sequenced on an Illumina HiSeq2500 next generation sequencer. Result: EdgeR analysis with significance cut-off of FDR<0.01 identified 629 genes that are significantly downregulated, and 779 genes that are significantly upregulated, in both the nonstop and sgf11 visual system glia as compared to wild-type glia. The active transcriptome (nuclear mRNA) of glial cells from wild type, nonstop and sgf11 were generated by deep sequencing of four biological replicates for each genotype using Illumina HISeq2500