Protocol for identifying glycosylated RNAs using solid-phase chemical conjugation and glycosidase release

RNA glycosylation plays a critical role in cellular signaling. Here, we present a protocol for enriching glycoRNAs from cultured cells and clinical tissues using a solid-phase chemoenzymatic technique. We describe steps for employing galactose oxidase (GAO) to selectively oxidize terminal sugar residues, enabling covalent capture on hydrazide resin, followed by enzymatic release using PNGase F. This scalable and robust assay efficiently enriches N-linked glycoRNAs, facilitating the systematic characterization of disease-specific RNA glycosylation patterns.