Drosophila melanogaster
Protocols: In brief, groups of 50 testes for different genotypes were ground in 750 μl of TRIzol reagent and incubated at room temperature for 10 min.  Phenol was removed from samples by multiple rounds of chloroform extraction. RNA from the supernatant was precipitated by adding 0.5x isopropanol and washed once with 70% ethanol. The extracted RNA was then treated with RNase-free DNase I (New England Biolabs, M0303) for 30 min at 37°C to remove DNA followed by heat-inactivating DNase I by incubating 10 min at 65°C upon adding 1 μl of 50 mM EDTA.  Approximately 500 ng of total RNA was used for library prep. Following DNase I digestion, long RNA (>200 nt) was isolated using Zymo Clean and Concentrator columns (Zymo, R1019). Subsequently, rRNA was removed using the NEBNext mRNA Magnetic Isolation Module (New England Biolabs, E7490L). With the purified RNA, cDNA libraries were prepared by employing the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs, E7760) and double-stranded cDNA was purified using AMPure XP Beads (Beckman Coulter, A63881).