Sequencing synthesized pre-miRNAs by iLIME-seq. (A) The diagram to show iLIME-seq. This is conducted using one RT and two PCR steps. The adapter and sequencing sequences are added in the PCR primers of the first and second PCR steps, respectively. (B) iLIME-seq was conducted for synthesized pre-miRNAs, and the reads resulting from NGS were mapped to the pre-miRNAs. The percentages of the perfect mapped reads were estimated for each pre-miRNA. (C) The sequencing results obtained after iLIME-seq were conducted for pre-mir-216a. (D) The quality of the synthesized uridylated pre-let-7a-1 variants were assessed via 12% urea-PAGE. (E) iLIME-seq was conducted on wild-type (WT) and six (1U–6U) uridylated pre-let-7a-1 variants. The percentage of accurate pre-miRNA sequences was estimated from the NGS results. Primer sequences used for pre-miRNA sequencing libraries are shown in Supplemental Table S2.
