Effects of Overexpression of Dube3a in Drosophila Brain

Fly Dube3a was expressed using either the neuronal driver (elav-GAL4) or the glial driver (Repo-GAL4). RNAseq data was compared among groups (elav>Dube3a), (repo>Dube3a) and (Dube3a alone). Expression was measured in whole dissected fly brain. Overall design: Flies expressing Dube3a (Das45) were grown at 25deg and aged approximately 3 days prior to brain dissections. RNA was isolated from control (uas-Dube3a alone), glial Dube3a overexpression (repo>Dube3a), or neuronal overexpression of Dube3a (elav>Dube3a) flies by removing 15-25 fly heads per genotype and homogenizing in TRI Reagent (Applied Biosystems). Total RNA was extracted with Directzol RNA MiniPrep Plus (Zymo Research Corp) according to the manufacturer's instructions which included a DNAse treatment step. Purity and concentration was verified using NanoDrop spectrophotometer (ThermoFisher Scientific), and integrity was confirmed using a Bioanalyzer (Agilent). Each genotype was run in triplicate. RNAseq was performed by PGM sequencing at the UTHSC Molecular Resource Center (MRC). Each RNA sample was converted to ion torrent sequencing libraries and run on 318v2 chips using an Ion Torrent Proton sequencer. All groups and replicates were run simultaneously to eliminate chip-to-chip variability.