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Ribocutter: Cas9-mediated rRNA depletion from multiplexed Ribo-seq libraries

View ORCID ProfileOscar G. Wilkins, View ORCID ProfileJernej Ule
doi: https://doi.org/10.1101/2021.07.14.451473
Oscar G. Wilkins
1Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK
2The Francis Crick Institute, London, UK
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  • ORCID record for Oscar G. Wilkins
Jernej Ule
1Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology, London, UK
2The Francis Crick Institute, London, UK
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  • For correspondence: jernej.ule@crick.ac.uk
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Abstract

RNA sequencing libraries produced from fragmented RNA, especially Ribo-seq libraries, contain high proportions of reads from abundant non-coding RNAs. Here, we describe a streamlined Cas9-based protocol for removing abundant rRNA/ncRNA contaminants from Ribo-seq (or other small RNA-seq) libraries and an easy-to-use software tool, ribocutter, for designing ready-to-order sgRNA templates. Following sgRNA template design, the pool of templates is in vitro transcribed using a 1-step commercial kit, which produces enough sgRNAs for multiple treatments. A single multiplexed sequencing library is then treated with Cas9/sgRNAs, followed by a short PCR program, which can increase the fraction of useful reads by more than 3-fold. Comparison of samples before and after depletion demonstrates that Cas9 produces minimal off-target effects and preserves key features (eg. footprint length, periodicity) of Ribo-seq libraries. The method is thus highly effective, costs <£0.50 per sample, and minimises non-specific depletion and technical variation between samples.

Competing Interest Statement

The authors have declared no competing interest.

Copyright 
The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC 4.0 International license.
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Posted July 14, 2021.
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Ribocutter: Cas9-mediated rRNA depletion from multiplexed Ribo-seq libraries
Oscar G. Wilkins, Jernej Ule
bioRxiv 2021.07.14.451473; doi: https://doi.org/10.1101/2021.07.14.451473
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Ribocutter: Cas9-mediated rRNA depletion from multiplexed Ribo-seq libraries
Oscar G. Wilkins, Jernej Ule
bioRxiv 2021.07.14.451473; doi: https://doi.org/10.1101/2021.07.14.451473

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