Ribocutter: Cas9-mediated rRNA depletion from multiplexed riboseq libraries

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 <{pound}0.50 per sample, and minimises non-specific depletion and technical variation between samples.