library_construction_protocol: Total RNA was extracted from 500 10-day-old adult Drosophila heads using TRIzol (Invitrogen, 15596026); RNA concentrations were measured on a Nanodrop 8000 spectrophotometer. Total RNA isolated from Drosophila heads was analyzed via RNA ScreenTape prior to ONT library generation; all samples achieved an RIN >9.8. 75 g of total RNA was enriched for full-length polyA RNA using the Dynabeads mRNA Purification kit (Invitrogen, 61005). For Illumina, 10 fly heads (five female and five male) per biological replicate were homogenized in radioimmunoprecipitation buffer (50 mM tris-HCL, 0.5% Nonidet-P40, 1.5 mM MgCl, 140 nM NaCl, and 1 mM dithiothreitol) with 1 μL (40 units/μL) of RNaseOUT (Invitrogen) and precleared with 40 μl of protein A Dynabeads (Invitrogen) for one hour at 4°C. After bead clearance, precleared lysate was split in two aliquots and diluted to a volume of 200 μl with cold binding buffer (0.0025% Triton X-100 in 1× PBS). Precleared lysate (200 μl) was stored at −80°C for RNA extraction via TRIzol. 500 ng polyA tailed RNA was used for library preparation using the Direct RNA Sequencing kit (SQK-RNA002) and protocol from ONT.RNA quality of the libraries was assessed using a Qubit 4 fluorometer (Invitrogen) prior to sequencing using the MinION Mk1C with R9.4 flow cells. For Illumina, RNA libraries from 0.7-1 μg of RNA were generated using the TruSeq Stranded mRNA Library Prep Kit and accompanying Index Set (Illumina Inc).