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CRISPR/Cas9-targeted removal of unwanted sequences from small-RNA sequencing libraries

Hardigan, Andrew A, Roberts, Brian S, Moore, Dianna E, Ramaker, Ryne C, Jones, Angela L, Myers, Richard M·2019·Nucleic Acids Research
DOI 10.1093/nar/gkz425PMC PMC6698666

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Abstract

In small RNA (smRNA) sequencing studies, highly abundant molecules such as adapter dimer products and tissue-specific microRNAs (miRNAs) inhibit accurate quantification of lowly expressed species. We previously developed a method to selectively deplete highly abundant miRNAs. However, this method does not deplete adapter dimer ligation products that, unless removed by gel-separation, comprise most of the library. Here, we have adapted and modified recently described methods for CRISPR/Cas9–based Depletion of Abundant Species by Hybridization (‘DASH’) to smRNA-seq, which we have termed miRNA and Adapter Dimer—DASH (MAD-DASH). In MAD-DASH, Cas9 is complexed with single guide RNAs (sgRNAs) targeting adapter dimer ligation products, alongside highly expressed tissue-specific smRNAs, for cleavage in vitro. This process dramatically reduces adapter dimer and targeted smRNA sequences, can be multiplexed, shows minimal off-target effects, improves the quantification of lowly expressed miRNAs from human plasma and tissue derived RNA, and obviates the need for gel-separation, greatly increasing sample throughput. Additionally, the method is fully customizable to other smRNA-seq preparation methods. Like depletion of ribosomal RNA for mRNA-seq and mitochondrial DNA for ATAC-seq, our method allows for greater proportional read-depth of non-targeted sequences.

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Metadata

Authors
Hardigan, A. A., Roberts, B. S., Moore, D. E., Ramaker, R. C., Jones, A. L., & Myers, R. M.
Affiliations
HudsonAlpha Institute for Biotechnology, Huntsville, AL 35806, USA
ORCID
Hardigan, Andrew A
Venue
Nucleic Acids Research
Publisher
Oxford University Press (OUP)
Published
2019-06-05
Volume
47
Issue
14
Pages
e84-e84
ISSN
  • 0305-1048
  • 1362-4962
DOI
10.1093/nar/gkz425
PMC
PMC6698666
Cited by
37
References
7
Language
en
Licence
creativecommons.org/licenses/by-nc/4.0/ (vor)
Funders
  • University of Alabama at Birmingham (TR0001417)
  • UAB Medical Scientist Training Program (5T32GM008361)

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