Sanger sequencing of Single cleavage product
The long cleavage products generated from the pri-miRNA cleavage assays were extracted and purified by IPA. The purified RNAs were ligated with a 5'-adapter (RA5-4N, GUU CAG AGU UCU ACA GUC CGA CGA UCN NNN) using T4 RNA ligase I (NEB, M0204L). The ligated RNAs were reverse transcribed using Superscript IV reverse transcriptase (Invitrogen, 18090050) and the RT primer specific to the 3'-segment of each pri-miRNA. The resulted cDNAs were PCR-amplified using a pair of sequencing primers, RP1 (AAT GAT ACG GCG ACC ACC GAG ATC TAC ACG TTC AGA GTT CTA CAG TCC GA), and each of the specific primers, RPIx. The DNAs were Sanger-sequenced using F-RP1 primer (AAT GAT ACG GCG ACC ACC GAG ATC TA) and specific reverse primer.