5 fluorescein 5 fam phosphoramidites

RNA oligonucleotides were synthesized according to previously established methods (B. Liu et al., 2018 (link)). In brief, modified (N⁶-methyladenosine (m⁶A), 5′-biotin, and 5′-fluorescein (5′-FAM)-labeled) and unmodified RNA oligonucleotides were synthesized with the MerMade 6 Oligo Synthesizer system, using 2′-tBDSilyl protected phosphoramidites (ChemGenes Corporation) on 1 μmol standard synthesis columns (BioAutomation). m⁶A phosphoramidites were obtained from ChemGenes. 5′-biotin phosphoramidites, and 5′-fluorescein (5′-FAM) phosphoramidites were obtained from Glen Research. Post-synthesis, oligonucleotides were cleaved and subjected to base deprotection, then quenched with Glen-Pak RNA quenching buffer and loaded onto Glen-Pak RNA cartridges (Glen Research) for purification according to the manufacturer’s instructions. Samples were then ethanol precipitated and buffer exchanged into nuclease-free water, then stored at −80°C until usage. DMT-off 2′O deprotected samples were directly ethanol precipitated and purified using large-scale 20% (w/v) denaturing TBE-PAGE, followed by electroelution into 20 mM Tris buffer, pH 8.0 and buffer exchange into nuclease-free water, then stored at −80°C until usage.