Optimized design of antisense oligomers for targeted rRNA depletion.
Optimized design of antisense oligomers for targeted rRNA depletion.
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DOI：
10.1093/nar/gkaa1072
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发表时间：
2021-01-11
影响因子：
14.9
通讯作者：
Lee MT
中科院分区：
文献类型：
--
作者：
Phelps WA;Carlson AE;Lee MT
RNA测序（RNA-seq）广泛用于定量全转录组的基因表达。虽然通常与多聚腺苷酸（poly（A））选择配对以富集信使RNA（mRNA），但许多应用需要替代方法来抵消总RNA中高比例的核糖体RNA（rRNA）。最近，使用RNaseH和平铺靶rRNA的反义DNA寡聚体的消化已经成为商业rRNA消耗试剂盒的替代方案。在这里，我们提出了一个流线型的，更经济的RNaseH介导的rRNA消耗与大幅降低前期成本，使用较短的反义寡核苷酸只有稀疏平铺沿着靶RNA在5分钟的消化反应。我们介绍了一种新的Web工具，Oligo-ASST，它简化了寡核苷酸设计，以最佳的热力学性质的目标区域，此外还可以生成紧凑的，共同的寡核苷酸池，同时靶向不同的RNA，例如在不同的物种。我们证明了这些策略的有效性，通过产生非洲爪蟾和斑马鱼，在胚胎发生过程中表达两种不同版本的rRNA的rRNA-耗尽寡核苷酸。所得的RNA-seq文库将rRNA减少到比对读数的<5%，与poly（A）选择相当，并且还揭示了许多非腺苷酸化RNA种类的表达。Oligo-ASST可在https://mtleelab.pitt.edu/oligo上免费获得，用于设计任何分类单元的反义寡核苷酸或靶向任何丰富的RNA进行消耗。Oligo-ASST设计优化的反义寡核苷酸组以靶向RNA用于耗尽。
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. Oligo-ASST designs optimized antisense oligo sets to target RNAs for depletion.
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影响因子：
7
作者：
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发表时间：
2014
影响因子：
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