To generate biotinylated circRNAs, in vitro transcription was conducted with T7 RNA polymerase (Takara, Dalian, China) and a biotin RNA labeling mix (Roche, Basel, Switzerland), using the recovered PCR product from the agarose gel as a template. The transcript product was ligated using an RNA ligase (Takara), and the ligated product was treated with DNase I and RNase R to generate biotinylated HBV_circ_1 and circ_GFP. The primer sequences T7-HBV_circ_1-Forward/ T7-HBV_circ_1-Reverse, and T7-circ_gfp-Forward/T7-circ_gfp-Reverse for in vitro circRNAs synthesis are listed in Table 1 .
Rna ligase
Manufactured by Takara Bio
Sourced in Japan
RNA ligase is an enzyme that catalyzes the formation of a phosphodiester bond between the 3' hydroxyl group of one RNA strand and the 5' phosphate group of another RNA strand. This process is essential for various RNA processing and modification reactions in cells.
Automatically generated - may contain errors
2 protocols using rna ligase
1
Biotinylated circRNA Synthesis
2
Identification and Validation of miR828 Targets
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The full-length cDNA sequence of pri-MIR828 was obtained by rapid amplification of cDNA ends (RACE)-PCR (Lai et al., 2012 (link)) using gene-specific primers (Supplementary Table S1 ) designed using the partial pri-MIR828 sequence (Suzuki et al., 2016 (link)) and total RNA isolated from Lollypop tepals. The amplified fragments were cloned into the pGEM-T Easy Vector (Promega, Tokyo, Japan) and sequenced [DNA DataBank of Japan (DDBJ) accession numbers LC569960 and LC569961]. The nucleotide sequences were aligned using the default parameters in Clustal W in the Kyoto University Bioinformatics Center website.1 RNA ligase mediated (RLM)-RACE PCR was carried out to validate the targets of miR828. RNA adapter and total RNA isolated from lily tepals or tobacco leaves were ligated using RNA ligase (Takara, Otsu, Japan). The ligated RNA was used for cDNA synthesis using the reverse primer of MYB12 and PrimeScript II reverse transcriptase (Takara). Nested PCR was performed using the two sets of primers. Sequences of the RNA adapter and primers are listed in Supplementary Table S1 . The amplified fragments of the appropriate size were cloned into the pMD20-T vector (Takara) and sequenced.
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