Mtp anchorchip 384 target plate

Captured miRNAs were purified with a Zip Tip C18 column (Millipore, Billerica, MA, USA) according to the manufacturer’s protocol. Purified samples were mixed with an aqueous solution of 3-hydroxypicolinic acid (Bruker Daltonics, Bremen, Germany) at a ratio of 1:1 (v/v) and applied to the target plate. One microliter of the mixture was applied to an MTP AnchorChip 384 target plate (Bruker Daltonics) and air-dried at room temperature. MALDI-TOF-MS analysis was performed with an ultrafleXtreme MALDI-TOF/TOF mass spectrometer (Bruker Daltonics) operated in negative ion and reflectron modes. Spectra were manually acquired using FlexControl software (v.3.3.108.0) (Bruker Daltonics). An analysis of the RNAs following hydrazine treatment revealed that the peaks contained m5C^{25 (link)}. Methylated adenines were determined to be 6 mA by sequential MS analysis following treatment with dimethylsulfate, which preferentially alkylates the N1 of adenines in RNA^{26 (link)}.

Total RNA was hybridized with single-strand DNA oligonucleotides complementary to miR-200c-5p (5′-CCA AAC ACT GCT GGG TAA GAC G-3′) that were adenosine-methylated at the 5′ end via a C6 linker purchased from Hokkaido System Science Co., Ltd. The mixture was heated to 95 °C, then gradually cooled to 30 °C to anneal miR-200c-5p and the complementary DNA. The miR-200c-5p–DNA complex was incubated with Dynabeads M-270 Amine (Cat. No. 14307D, ThermoFisher Scientific) at 4 °C for 1 h. The mixture was heat-eluted and the supernatant obtained by magnetic separation. Lyophilized samples were used for subsequent experiments. Captured miR-200c-5p was purified using a ZipTip C₁₈ cartridge column (Cat. No. ZTC18M96; MilliporeSigma) according to the manufacturer’s protocol. Purified miR-200c-5p was mixed with an aqueous solution of 3-hydroxypicolinic acid (Cat. No. 8201224; Bruker Daltonics) in a 1:1 (v/v) ratio, and 1 µl of the mixture was applied to an MTP AnchorChip 384 target plate (Cat. No. 8209514; Bruker Daltonics) and air-dried at room temperature. MALDI–TOF MS/MS analysis was performed using an ultrafleXtreme MALDI–TOF mass spectrometer (Bruker Daltonics) operated in negative-ion and reflectron modes. Spectra were manually acquired by FlexControl software v. 3.3.108.0 (Bruker Daltonics).