Transcriptomes of the developing larval ovaries of Drosophila melanogaster during terminal filament formation

Insect ovaries are made up of tubular egg producing subunits called ovarioles, whose number largely determines the number of eggs that can be potentially laid. Ovariole number is directly determined by the number of cellular structures called terminal filaments, which are stacks of cells that assemble in the larval ovary. Elucidating the developmental and regulatory mechanisms of terminal filament formation is thus key to understanding the regulation of insect reproduction through ovariole number regulation. We systematically measured mRNA expression of all cells in the Drosophila larval ovary at the beginning, middle and end of terminal filament formation using RNA-seq. We also separated somatic and germ line cells during these stages and assessed their tissue-specific gene expression during larval ovary development. We extracted RNA from the whole ovaries of Drosophila melanogaster larvae at three stages - the beginning, middle and end of terminal filament formation. The three larval developmental stages were assigned as follows- early (72 hours After Egg Laying, h (AEL)), middle (96h AEL) and late (120h AEL) stages. In a subsequent experiment we used Fluorescence Activated Cell Sorting (FACS) to separate GFP-expressing somatic (bab Gal4>GFP) or germ line (nos Gal4>GFP) tissues of the three stages of larval ovaries. Total RNA was isolated from the three stages of whole ovaries and the two tissue types. Three biological replicates of cDNA libraries were prepared for each sample from Poly-A enriched-RNA using Takara PrepX kit. These cDNA libraries were sequenced using a single end 50bp protocol in an Illumina HiSeq 2500 sequencer. Sequences obtained were checked for quality and replicates with 10 million or more reads were considered for downstream differential expression analysis.