Self-cleaving ribozymes are catalytic RNAs and can be found in all domains of life. They catalyze a site-specific cleavage that results in a 5′ fragment with a 2′,3′ cyclic phosphate (2′,3′ cP) and a 3′ fragment with a 5′ hydroxyl (5′ OH) end. Recently, several strategies to enrich self-cleaving ribozymes by targeted biochemical methods have been introduced by us and others. Here, we develop an alternative strategy in which 5ʹ OH RNAs are specifically ligated by RtcB ligase, which first guanylates the 3′ phosphate of the adapter and then ligates it directly to RNAs with 5′ OH ends. Our results demonstrate that adapter ligation to highly structured ribozyme fragments is much more efficient using the thermostable RtcB ligase from Pyrococcus horikoshii than the broadly applied Escherichia coli enzyme. Moreover, we investigated DNA, RNA and modified RNA adapters for their suitability in RtcB ligation reactions. We used the optimized RtcB-mediated ligation to produce RNA-seq libraries and captured a spiked 3ʹ twister ribozyme fragment from E. coli total RNA. This RNA-seq-based method is applicable to detect ribozyme fragments as well as other cellular RNAs with 5ʹ OH termini from total RNA.

MONSDA runs HTS data analysis from pre- to postprocessing based on a single configuration file. It wraps Snakemake or Nextflow to run workflow steps involving QC, trimming, mapping, deduplication and differential analysis as well as a set of specific workflows for the generation of genome browser tracks and more. Users profit from many of the advantages of two of the most popular workflow management systems without having to learn all the specifics of Snakemake or Nextflow and can interchange and configure tools according to their specific needs. MONSDA is available via Bioconda, pip and https://github.com/jfallmann/MONSDA