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

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.

journal_name

Nucleic Acids Res

journal_title

Nucleic acids research

authors

Hardigan AA,Roberts BS,Moore DE,Ramaker RC,Jones AL,Myers RM

doi

10.1093/nar/gkz425

subject

Has Abstract

pub_date

2019-08-22 00:00:00

pages

e84

issue

14

eissn

0305-1048

issn

1362-4962

pii

5511469

journal_volume

47

pub_type

杂志文章

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