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. Overall design: 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