Optimized design of antisense oligomers for targeted rRNA depletion.

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    • Source:
      Publisher: Oxford University Press Country of Publication: England NLM ID: 0411011 Publication Model: Print Cited Medium: Internet ISSN: 1362-4962 (Electronic) Linking ISSN: 03051048 NLM ISO Abbreviation: Nucleic Acids Res Subsets: MEDLINE
    • Publication Information:
      Publication: 1992- : Oxford : Oxford University Press
      Original Publication: London, Information Retrieval ltd.
    • Subject Terms:
    • Abstract:
      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.
      (© The Author(s) 2020. Published by Oxford University Press on behalf of Nucleic Acids Research.)
    • Comments:
      Erratum in: Nucleic Acids Res. 2021 Nov 8;49(19):11406. (PMID: 34591961)
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    • Grant Information:
      R01 GM125638 United States GM NIGMS NIH HHS; R35 GM137973 United States GM NIGMS NIH HHS
    • Accession Number:
      0 (Oligodeoxyribonucleotides, Antisense)
      0 (RNA, Messenger)
      0 (RNA, Ribosomal)
      24937-83-5 (Poly A)
      63231-63-0 (RNA)
      EC 3.1.26.4 (Ribonuclease H)
    • Publication Date:
      Date Created: 20201122 Date Completed: 20210201 Latest Revision: 20210930
    • Publication Date:
      20240829
    • Accession Number:
      PMC7797071
    • Accession Number:
      10.1093/nar/gkaa1072
    • Accession Number:
      33221877