Optimized identification and characterization of small RNAs with PANDORA-seq.

Small noncoding RNAs (sncRNAs) are a diverse group of RNAs including small interfering RNAs, microRNAs, PIWI-interacting RNAs and RNAs derived from structured RNAs such as transfer RNAs, ribosomal RNAs and others. These sncRNAs have varied termini and RNA modifications, which can interfere with adaptor ligation and reverse transcription during cDNA library construction, hindering detection of many types of sncRNA by standard small RNA sequencing methods. To address this limitation, PANDORA sequencing introduces a refined methodology. The procedure includes sequential enzymatic treatments of size-selected RNAs with T4PNK and AlkB, which effectively circumvent the challenges presented by the ligation-blocking termini and reverse transcription-blocking RNA modifications, followed by tailored small RNA library construction protocols and deep sequencing. The obtained datasets are analyzed with the SPORTS pipeline, which can comprehensively analyze various types of sncRNA beyond the traditionally studied classes, to include those derived from various parental RNAs (for example, from transfer RNA and ribosomal RNA), as well as output the locations on the parental RNA from which these sncRNAs are derived. The entire protocol takes ~7 d, depending on the sample size and sequencing turnaround time. PANDORA sequencing provides a transformative tool to further our understanding of the expanding small RNA universe and to explore the uncharted functions of sncRNAs.