RNA-Seq of Drosophila melanogaster: adult female head
All the flies used in this study were from Bloomington Drosophila Stock Center. They were reared at 25 C in vials or bottles containing standard agar-cornmeal-sugar-yeast-Tegosept media.  The virgins of the different RNAi strains were crossed with males of the desired driver strains. After approximately  six days of mating, we discarded the parental flies and transferred F1 offspring to fresh food. We extracted RNA from testes using Quick-RNA Plus Kit (Zymo Research R1057) using 20-80 pairs of testes from 3-7-day old mated males. The testes were dissected in 1X PBS and immediately transferred into 250 L RNAlater Solution. Add 250 L ice-cold 1X PBS, and centrifuge at 5000g for 1 min at 4 C. After removing the supernatant and adding 300 L 1x DNA/RNA Shield, they were immediately homogenized using an electric pestle. We then followed the kit protocol to isolate purified RNAs >200 nt using columns with DNase I treatment. We extracted RNA from heads using TRIzol/chloroform followed by Zymo RNA Purification. We used heads of 80 3-7-day old mated females. The flies were collected by freezing and heads were isolated on dry ice. We homogenized heads in 500 L TRIzol Reagent, and then added 100 L chloroform. We collected the upper aqueous layer after spinning, and added the same volume of 100% ethanol, and then purified the RNA using Quick-RNA Plus Kit (Zymo Research R1057) with DNase I treatment. We prepared ribo-zero RNA using siTools rRNA depletion Kit from Galen Laboratory Supplies (dp-P020-000007). Starting with as much as possible total RNA up to 5 g, we followed the kit protocols to remove abundant ribosomal RNAs. Thermo Fisher MyOne Streptavidin C1 Dynabeads (#65001) were used. We purified and concentrated ribo-zero RNA using RNA Clean & Concentrator-5 kit from Zymo Research (R1015). Using all available ribo-zero RNA, we purified RNAs >200 nt.  We prepared total RNA-seq libraries using NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (E7760). Starting with 1 ng-100 ng purified ribo-zero RNA, follow the kit protocols to carry out RNA fragmentation and priming (7 min at 94 C), first strand cDNA synthesis (incubation for 45 min at 42 C), second strand cDNA synthesis, purification of Double-stranded cDNA, end prep of cDNA library, adaptor ligation, purification of the ligation reaction, PCR enrichment of adaptor ligated DNA, and twice purification of PCR reaction.