GSM9290598: wtOSC_rRNA-depleted_RNAseq; Drosophila melanogaster; RNA-Seq
Total RNA was isolated using Trizol (Invitrogen) according to the manufacturer's instructions. Ribosomal RNA was depleted using an RNAseH based protocol(Morlan, Qu et al. 2012, Adiconis, Borges-Rivera et al. 2013). In brief, standard 50 mer DNA oligos complementary to rRNA precursor and mitochondrial rRNA sequences were annealed to 1 µg total RNA in Hybridase buffer with EDTA. Samples were denatured (95 °C, 3 min), cooled gradually to 45 °C, and incubated with Thermostable Hybridase RNAseH (1 h, 45 °C) to specifically degrade rRNA. Following digestion, samples were treated with Turbo DNAse (30 min, 37 °C) and purified using RNA Clean & Concentrator 5 columns (Zymo). Elution was carried out in NEBNext Ultra II First Strand Synthesis buffer for downstream library preparation. RNA seq libraries were generated using the NEBNext Ultra II 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.