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

We performed this analysis on pre-miRNA-seq, miRNA-seq, and smRNAseq (without IP) from human HEK293T cells as well as pre-miRNA-seq and mature miRNA-seq data previously generated using the same technique on MEFs (19) . We successfully aligned 10.8% of pre-miRNA-seq, 98.7% of miRNAseq, and 52.4% of smRNA-seq reads to the human miRBase v20 annotation set (Figs. 1D, S1A, and Table S1 ). We were not surprised to find such high rates of mapping for mature miRNAs, however, a 10% mapping rate for pre-miRNAs is significantly higher than previous attempts to sequence pre-miRNAs without pre-miRNA specific primers (0.8%) (27) . Using the datasets previously generated from MEFs, we had a much higher rate of pre-miRNA-seq reads mapping (44%), but mapped fewer miRNA-seq reads (90.3%) ( Fig. S2A and Table S1 ). These differences likely represent variable experimental conditions and amounts of starting material and/or biological differences in the smRNA populations between these two mammals.