EFFICIENT RIBOSOMAL RNA DEPLETION FROM DROSOPHILA TOTAL RNA FOR NEXT-GENERATION SEQUENCING APPLICATIONS

We developed a cost-effective enzyme-based rRNA-depletion method tailored for Drosophila melanogaster, addressing the limitations of existing commercial kits and the lack of peer-reviewed alternatives. Our method employs single-stranded DNA probes complementary to Drosophila rRNA, forming DNA-RNA hybrids. These hybrids are then degraded using RNAse H enzyme, effectively removing rRNA and enriching all non-ribosomal RNAs, including mRNA, lncRNA and small RNA. When compared to a commercial rRNA Fly Kit, our approach demonstrated superior rRNA removal efficiency and mapping percentage, confirming its effectiveness. Additionally, our method successfully enriched the non-coding transcriptome, making it a valuable tool for studying ncRNA in Drosophila. The probe sequences and rRNA-depletion protocol is made freely available, offering researchers a reliable alternative for rRNA-depletion experiments. Overall design: RNA-seq profiling of Drosophila adult brains processed by PolyA enrichment and rRNA depletion

针对现有商用试剂盒的局限性以及缺乏经过同行评议的替代方案这一问题，我们开发了一款专为黑腹果蝇（Drosophila melanogaster）设计的经济高效的酶法核糖体RNA（rRNA）去除方法。本方法采用与黑腹果蝇核糖体RNA互补的单链DNA探针，形成DNA-RNA杂交双链。随后利用核糖核酸酶H（RNase H）降解这些杂交产物，可有效去除核糖体RNA，并富集所有非核糖体RNA，包括信使RNA（messenger RNA）、长链非编码RNA（long non-coding RNA）以及小型RNA。与商用果蝇核糖体RNA去除试剂盒（rRNA Fly Kit）相比，本方法展现出更优异的核糖体RNA去除效率与比对率，证实了其应用有效性。此外，本方法可成功富集非编码转录组，成为研究黑腹果蝇中非编码RNA（non-coding RNA）的极具价值的工具。本方法所涉及的探针序列与核糖体RNA去除实验方案已免费公开，可为研究人员开展核糖体RNA去除相关实验提供可靠的替代选择。实验整体设计：对经聚腺苷酸（PolyA）富集与核糖体RNA去除处理的黑腹果蝇成体脑组织进行RNA测序（RNA-seq）谱分析。