Synthesis of pri-miRNA from C. elegans

Each DNA sequence coding for pri-miRNAs contained a DNA sequence coding for pre-miRNA and its 30 bp upstream and 25 bp downstream sequence in the C.elegans genomic DNA. Each pri-miRNA-coding DNA was added with a T7 promoter sequence at its 5p-end to generate a DNA template for in vitro transcription (IVT), so-called IVT-DNAs. The IVT-DNAs were synthesized by 2 methods. Twenty IVT-DNAs were produced by Phusion™ Hot Start II DNA Polymerase (Thermo Scientific) in the PCR reaction using a pair of primers and C.elegans genomic DNA or a synthetic ssDNA backbone as PCR template. One hundred and seventeen IVT-DNAs were made by Klenow Fragment (Thermo Scientific), which extended the partial dsDNAs generated from annealing pairs of synthetic ssDNAs. The primer sequences and synthesis methods for each IVT-DNA are shown in Supplementary Table S2. The pri-miRNAs were synthesized in a 20 μl IVT reaction mixture containing 200 ng IVT-DNA using the MEGAscript T7 Kit (Invitrogen). The IVT mixture was incubated at 37°C for 12 h. The IVT-DNA templates were then digested using TURBO DNase (Thermo Scientific). The reaction mixture was treated with 20 μl 2x TBE-Urea buffer and denatured at 75°C for 5 min. The denatured RNA was loaded onto a pre-run 10% Urea-PAGE. The RNA at the expected size was gel-purified and air-dried. Finally, the RNA was dissolved in distilled water and stored at -80°C for later use.