mRNA was first heated at 85°C for 5 min and chilled on ice for 2 min to denature RNA secondary structure. The FTO demethylation reaction was conducted before MazF reaction in a 20-μl reaction mixture containing 100 to 200 ng mRNA, 2.5 μg of FTO demethylase, 283 μM (NH4)2Fe(SO4)2∙6H2O, 300 μM α-KG, 2 mM l-ascorbic acid, 20 U of RNase inhibitor, and 50 mM tris-HCl buffer (pH 7.5). After incubating at room temperature for 3 hours, the reaction was stopped by adding 40 mM EDTA. To prevent the influence of FTO buffer, the MazF treatment was supplemented with the same reaction mixture except FTO protein. The MazF and FTO-MazF samples were heated at 85°C for 5 min, chilled on ice for 2 min, and then incubated with 10 U of MazF in the MazF reaction buffer at 37°C for 30 min. The fragmented mRNA was purified by an RNA Clean & Concentrator-5 kit (Zymo Research) and eluted into 20 μl of RNase-free water. The eluted RNA fragment was end repaired by the T4 polynucleotide kinase (T4PNK; Vazyme Biotech) in 50-μl reaction mixture with 1× T4PNK buffer and incubated at 37°C for 30 min. After purification by an RNA Clean & Concentrator-5 kit (Zymo Research), the concentration of fragmented mRNA was measured by the Qubit RNA HS Assay Kit (Thermo Fisher Scientific). The NGS library was constructed by using the VAHTS Small RNA Library Prep Kit (Vazyme Biotech, NR801) with 10 ng of fragmented mRNA and amplified by PCR for 15 cycles. Libraries were loaded on the 8% urea polyacrylamide gel and electrophoresed in 0.5× TBE buffer, and purified libraries were sequenced on the Illumina HiSeq X10 platform. Three replicates have been conducted for mRNA of HEK293T cell line. The same protocol was also conducted for mammalian tissues.

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