title: GSM8647236: Pupal Wings at 24 after puparium formation (APF) of genotype rn-Gal4; UAS-brRNAi; UAS-E93RNAi rep1; Drosophila melanogaster; RNA-Seq
library_construction_protocol: A minimum of 40 pupal wings were dissected in 1× PBS from the following genotypes at 24 and 44 hours APF: rn-Gal4 (control), rn-Gal4; UAS-E93RNAi and rn-Gal4; UAS-brRNAi; UAS-E93RNAi. Pupal wings were then transferred to ice for subsequent RNA extraction using the Roche High Pure RNA Tissue Kit (Cat# 12033674001). Libraries were prepared using NEBNext Poly(A) mRNA Magnetic Isolation Module (ref. E7490, New England Biolabs) and NEBNext Ultra II Directional RNA Library Prep Kit for Illumina (ref. E7760) according to the manufacturer's protocol, to convert total RNA into a library of template molecules of known strand origin and suitable for subsequent cluster generation and DNA sequencing. Briefly, 250 ng of total RNA was used for polyA‐mRNA selection using Oligo‐dT beads, performing two rounds of purification. During the second elution of the mRNA, this was fragmented under elevated temperature and primed with random hexamers for cDNA synthesis. Immediately afterward, second strand cDNA was synthesized, incorporating dUTP in place of dTTP. dsDNA was then subjected to end repair and A‐tailing in a single reaction, and subsequent ligation of the NEB adapter. USER enzyme was used to excise the hairpin‐like adaptors. Finally, PCR selectively enriched those DNA fragments that had adapter molecules on both ends. The PCR was performed using NEBNext Multiplex Oligos for Illumina (96 Unique Dual Index Primer Pairs, ref. E6440, E6442, E6444, E6446) and the master mix provided with the kit. All purification steps were performed using AgenCourt AMPure XP beads (ref. A63882, Beckman Coulter). Final libraries were analyzed using Bioanalyzer DNA 1000 or Fragment Analyzer Standard Sensitivity (ref. 5067‐1504 or ref. DNF‐473, Agilent) to estimate the quantity and validate the size distribution, and were then quantified by qPCR using the KAPA Library Quantification Kit KK4835 (ref. 07960204001, Roche). Libraries were sequenced 1 * 51 + 10 + 10 bp on Illumina's NextSeq2000.