PT  - JOURNAL ARTICLE
AU  - Wilkins, Oscar G.
AU  - Ule, Jernej
TI  - Ribocutter: Cas9-mediated rRNA depletion from multiplexed Ribo-seq libraries
AID  - 10.1101/2021.07.14.451473
DP  - 2021 Jan 01
TA  - bioRxiv
PG  - 2021.07.14.451473
4099  - http://biorxiv.org/content/early/2021/07/14/2021.07.14.451473.short
4100  - http://biorxiv.org/content/early/2021/07/14/2021.07.14.451473.full
AB  - 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 StatementThe authors have declared no competing interest.