Isolation and sequencing of AGO-bound RNAs reveals characteristics of mammalian stem-loop processing in vivo

Ian M. Silverman; Sager J. Gosai; Nicholas Vrettos; Shawn W. Foley; Nathan D. Berkowitz; Zissimos Mourelatos; Brian D. Gregory

Using this approach, we successfully generated high-throughput sequencing libraries for both pre-miRNAs and mature miRNAs. We obtained 27.8 and 9.8 million reads with sufficient adapter sequence from the pre-miRNA-seq and mature miRNA-seq libraries, respectively (Table S1). Given that pre-miRNAs and mature miRNAs contain non-templated modifications at their 3' ends, we reasoned that standard alignment pipelines would be limited in their ability to align these sequences to miRBase. Therefore, we developed an alignment pipeline that utilized the first 35 nt of the pre-miRNA-seq (18 nt for miRNA-seq) reads for alignment (Fig. 1C) , followed by Smith-Waterman local alignment to extend the read as far as possible along the mapped miRNA sequence (see Methods) (43) (44) (45) . After all possible nucleotide matches were made, we selected . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder. It . https://doi.org/10.1101/294488 doi: bioRxiv preprint alignments with the lowest mismatch rate and captured non-templated modifications at the 3' end.