Biotin-tagged miRNA and rRNA Synthesis

The DNAs containing T7 RNA polymerase promoter and pri-miR-9-1 (137nt and 413nt) or pri-miR-29b-1 (144nt) (T7-RNA-polymerase-promoter-pri-miR-9-1/-pri-miR-29b-1) and helix 45 of 18S-rRNA (50nt) (T7-RNA-polymerase-promoter-18S-rRNA-45h) were separately amplified by PCR (Supplementary Table S1) from CD513B-pri-miR-9-1, CD513B-pri-miR-29b-1 and total RNA reverse transcribed cDNA. The 18S-rRNA-45h, pri-miR-9-1 and pri-miR-29b-1 RNAs were internally tagged with biotin by using Bio-16-UTP as well as ATP, UTP, CTP and GTP through T7 RNA polymerase transcription. Meanwhile, ac4C-pri-miR-9-1, ac4C-pri-miR-29b-1 and ac4C-18S-rRNA-45h RNAs were transcribed with ATP, UTP, GTP and N4-actylcytidine triphosphate (ac4CTP) as well as internally tagged with biotin by using Bio-16-UTP through T7 RNA polymerase transcription. Finally, all the transcribed RNAs were further purified from 8 M Urea/ 15% PAGE gel.