Duplexed Direct RNA Sequencing Protocol Using Polyadenylation and Polyuridylation

Oxford Nanopore Technologies provides multiplexing options for DNA and cDNA sequencing, but not for direct RNA sequencing. We use a duplex approach involving polyadenylation of one sample and polyuridylation of another to simultaneously sequence the native ribosomal RNA of two Saccharomyces cerevisiae strains containing a known differential RNA modification for downstream demultiplexing using bioinformatics approaches.