GSM9533208: siLUC_RZ_Rep1; Drosophila melanogaster; RNA-Seq
Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. RNA was treated with DNase I (ThermoFisher Scientific) and purified using RCC25 kit (Zymo Research) with on-column Zymo DNaseI according to the manufacturer's instructions. Eluted RNA was poly(A)-selected twice using magnetic Dynabeads Oligo (dT)25 (ThermoFisher Scientific). Libraries were  generated using the NEBNext Ultra Directional RNA Library Prep Kit (New England Biolabs) according to the manufacturer's instructions, with the exception of the final amplification. Libraries were PCR amplified with KAPA HiFi polymerase and barcoded Illumina primers, with amplification monitored in real time using EvaGreen fluorescence. Reactions were stopped once predetermined RFU thresholds were reached to avoid over amplification. Libraries were then purified, quantified by Qubit, assessed for fragment size, and multiplexed for Illumina sequencing.