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

Overall, we obtained pre-miRNA-seq reads mapping to 367 annotated human microRNAs and 267 annotated mouse microRNAs (Table S2) . For libraries prepared from smaller RNA species, we mapped reads to 931 (HEK239T miRNA-seq), 567 (MEF miRNAs-seq), and 1,364 (HEK293T smRNAseq) miRBase miRNAs. We examined the size distribution of mapped pre-miRNA-seq reads and found that they were broadly distributed between 55-65 nt in both cell types (Figs. 1E and S2B). In contrast, miRNA-seq reads, were tightly . CC-BY 4.0 International license is made available under a The copyright holder for this preprint (which was not peer-reviewed) is the author/funder.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 distributed between 21-24 nt, in both HEK293T AGO-bound and total cellular fractions, as well as in MEFs (Figs. 1F, S1B, and S2C). We determined the abundance, end concordance, and non-templated additions for all mapped miRNAs and generated coverage plots to represent these data, which are available for download at http://gregorylab.bio.upenn.edu/AGO_IP_Seq/ (Table S2 ; examples in Figs. 1G-H). Together, our biochemical and bioinformatic approaches provide a data rich resource for the global and unbiased analysis of pre-miRNAs in two mammals.