Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA 15260, USA.
Nucleic Acids Res. 2021 Jan 11;49(1):e5. doi: 10.1093/nar/gkaa1072.
RNA sequencing (RNA-seq) is extensively used to quantify gene expression transcriptome-wide. Although often paired with polyadenylate (poly(A)) selection to enrich for messenger RNA (mRNA), many applications require alternate approaches to counteract the high proportion of ribosomal RNA (rRNA) in total RNA. Recently, digestion using RNaseH and antisense DNA oligomers tiling target rRNAs has emerged as an alternative to commercial rRNA depletion kits. Here, we present a streamlined, more economical RNaseH-mediated rRNA depletion with substantially lower up-front costs, using shorter antisense oligos only sparsely tiled along the target RNA in a 5-min digestion reaction. We introduce a novel Web tool, Oligo-ASST, that simplifies oligo design to target regions with optimal thermodynamic properties, and additionally can generate compact, common oligo pools that simultaneously target divergent RNAs, e.g. across different species. We demonstrate the efficacy of these strategies by generating rRNA-depletion oligos for Xenopus laevis and for zebrafish, which expresses two distinct versions of rRNAs during embryogenesis. The resulting RNA-seq libraries reduce rRNA to <5% of aligned reads, on par with poly(A) selection, and also reveal expression of many non-adenylated RNA species. Oligo-ASST is freely available at https://mtleelab.pitt.edu/oligo to design antisense oligos for any taxon or to target any abundant RNA for depletion.
RNA 测序（RNA-seq）广泛用于定量全转录组基因表达。尽管通常与多聚腺苷酸（poly(A)）选择结合以富集信使 RNA（mRNA），但许多应用需要替代方法来对抗总 RNA 中高比例的核糖体 RNA（rRNA）。最近，使用 RNaseH 和反义 DNA 寡核苷酸靶向 rRNA 的消化已成为商业 rRNA 耗尽试剂盒的替代方法。在这里，我们提出了一种简化、更经济的 RNaseH 介导的 rRNA 耗尽方法，使用更短的反义寡核苷酸，仅在 5 分钟的消化反应中稀疏地靶向目标 RNA，成本大大降低。我们引入了一种新颖的 Web 工具，Oligo-ASST，它简化了针对具有最佳热力学特性的区域的寡核苷酸设计，并且还可以生成紧凑、常见的寡核苷酸池，同时靶向不同的 RNA，例如不同的物种。我们通过为非洲爪蟾和斑马鱼生成 rRNA 耗尽寡核苷酸来证明这些策略的有效性，斑马鱼在胚胎发生过程中表达两种不同的 rRNA 版本。所得的 RNA-seq 文库将 rRNA 减少到对齐读数的<5%，与 poly(A) 选择相当，并且还揭示了许多非腺苷酸化 RNA 物种的表达。Oligo-ASST 可免费在 https://mtleelab.pitt.edu/oligo 上获得，用于设计任何分类群的反义寡核苷酸或靶向任何丰富的 RNA 进行耗尽。
