Catch and release of sialoglycoRNAs enables sequencing-based profiling across cellular and extracellular material

Glycosylated RNAs (glycoRNAs) represent a recently discovered class of small RNAs, but their systematic characterization has been limited by reliance on metabolic chemical reporters and high RNA input requirements. Here we present rPAL sequencing (rPAL-seq), a sensitive and selective platform for de novo discovery of sialoglycoRNAs. rPAL-seq combines enhanced periodate oxidation of sialic acids with a capture–release workflow and optimized library construction using poly(A) extension coupled with template-switching reverse transcription. The method enabled reproducible profiling from less than 100 ng of input RNA, corresponding to less than 2% of the material required by previous approaches. When applied across 13 human cell lines, rPAL-seq identified lineage-associated glycoRNA patterns alongside a conserved core dominated by uridine-rich snRNAs and snoRNAs, with modification signatures implicating glycosylation on acp³U or related uridine-based modifications. Extending to extracellular fractions, rPAL-seq revealed secreted glycoRNA profiles distinct from those of the cellular fraction. rPAL-seq provides a robust, scalable strategy for glycoRNA profiling, opening new avenues for studying this emerging biopolymer. Overall design: This study aimed to profile sialoglycoRNA from diverse human and animal cell lines using rPAL-seq, a sequencing method first introduced in this study that combines enhanced reductive periodate–aminoxy labeling with sialidase-specific elution. Cells (including HeLa, A549, 293T, Huh7, LPS853, LN308, OCI-AML2, OCI-AML3, Molm13, Jurkat, Jeko1, Nalm6, DiFi, MDCK, and EMT6) were cultured under standard conditions. Extracellular vesicles (EVs) and non-vesicular extracellular nanoparticles (NVEPs) were isolated from conditioned medium of DiFi and MDCK using previously established protocols. For each biological sample, sialoglycoRNA was captured and processed in 3 parallel groups: IP (VC sialidase–eluted), Control (mock elution with heat-inactivated enzyme), and Input (1% reserved prior to elution). Libraries were prepared using the rPAL-seq workflow and sequenced on an Illumina NovaSeq X platform with single-end 100 bp reads.