title: adult whole fly,dec2-SS, biol rep 1, nanopore long reads
design: Fly stocks were raised at 25oC with 12:12h L:D cycle and fed on a standard cornmeal molasses medium. The  fly strains were obtained from the Bloomington Drosophila Stock Center (BDSC): GMR23E10-gal4 (49032), UAS-dec2WT (64227), UAS-dec2P384R (64228). Drosophila melanogaster were reared on standard cornmeal molasses meduim, under controlled environment with temperature of 25oC and L:D cycle of 12:12. Flies for RNA extraction were collected at ZT 3, the time in which we observed the most significant difference in their daytime sleep. RNA was extraction from whole animal (~ 70 flies) using standard a TRIzol TM reagent protocol (Thermo Fisher Scientific, cat# 15596018) for both Illumina and nanopore sequencing. For illumina sequencing:  After RNA extraction, genomic DNA was removed using a GeneJet RNA-purification kit (Thermo Fisher Scientific, cat# K0702). RNA quality was measured using a Bioanalyzer and around 1.5-2 g of purified RNA with RIN above 6 for each replicates was sent to Novogene (Sacramento, CA) in dry ice. Novogene prepared cDNA library using polyA enrichment library preparation procedure  and then performed Illumina sequencing (Illumina NovaSeq 6000 PE 150).  For nanopore: 10 g of total RNA was used to prepare mRNA using NEXTflex Poly(A) Beads (BIOO Scientific cat # NOVA-512980). mRNA was assessed using a Bioanalyzer. 200ng of input mRNA was used to prepare cDNA libraries using a Direct cDNA sequencing (SQK-DCS109) kit and these were prepared according to the Oxford Nanopore recommended protocol. Around 60ng total cDNA libraries were sequenced on a MinION using R9.4 flow cells. For each genotype, three independent biological replicates were sequenced. 1.5-2 g of purified RNA  was sent to Novogene (Sacramento, CA). Novogene prepared cDNA library using polyA enrichment library preparation procedure to performed Illumina sequencing (Illumina NovaSeq 6000 ). For nanopore: 200ng of mRNA was used to prepare cDNA libraries using a Direct cDNA sequencing (SQK-DCS109) kit following Oxford Nanopore recommended protocol. Around 60ng total cDNA libraries were sequenced on a MinION using R9.4 flow cells. For each genotype, three independent biological replicates were sequenced.