RNA oligos (10 pmol) were incubated with 2.5 U of MazF (mRNA intereferase MazF, Takara Bio Inc., code no. 2415A) in the 20-μl reaction mixture of MazF buffer [40 mM sodium phosphate (pH 7.5) and 0.01% Tween 20] at 37°C for 30 min. The RNA oligos were mixed with the m6A percentage of 0, 20, 40, 60, 80, and 100%, and the oligo mixtures were incubated with 2.5 U of MazF in the 20-μl reaction mixtures. The samples were loaded into 15% urea polyacrylamide gel and electrophoresed in 0.5× tris-boric acid–EDTA (TBE) buffer.

The FTO demethylation reaction was conducted in the reaction mixture that contained 10 pmol of m6A RNA oligo, 0 to 10 μg of FTO demethylase, 283 μM (NH4)2Fe(SO4)2∙6H2O, 300 μM α-ketoglutarate (α-KG), 2 mM l-ascorbic acid, 20 U of RNase inhibitor (Takara Bio Inc., code no. 2313A), and 50 mM tris-HCl buffer (pH 7.5). The reaction was stopped by 40 mM EDTA after 3 hours of incubation at room temperature. The MazF cleavage reaction was performed to test the efficiency of FTO demethylation.

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