Analysis of GlycoRNAs in Glioma Cell Lines LN229 and U87

A number of studies have identified a novel class of biomolecules, namely glycosylated RNAs (glycoRNAs), which may play important roles in cellular physiology, pathology and immunity. However, whether glycoRNAs are present in glioma cells and what their functions are remain unanswered. In this study, through N-Azidoacetylmannosamine-tetraacylated (Ac4ManNAz) labeling method, glycoRNAs were enriched and purified from glioma cell lines LN229 and U87. Northern blot was performed to test the presence of glycoRNAs. It is noteworthy that we developed a sequencespecific RNA capture magnetic bead system to enrich for a specific glycoRNA such as U2, U4, and Y5. The components of glycoRNAs were analyzed and identified through RNA sequencing and chromatography-mass spectrometry. Overall design: We sought to analyze the composition of glycoRNAs enriched in glioma cells. Through a series of processes including Ac4ManNAz labelling, small RNA extraction, DBCObiotin treatment, purification by streptavidin magnetic beads and Zymo column, glycoRNAs from U87, LN229 and Hela cells were purified and used for small RNA deep sequencing analysis. RNA samples from U87, LN229 and Hela cells treated with Ac4ManNAz were designated U87-24h, LN229-24h and Hela-24h, respectively. RNA samples from U87, LN229 and Hela cells without Ac4ManNAz treatment served as inputs and were designated U87-0h, LN229-0h and Hela-0h, respectively. The analysis of small RNA expression revealed significant differences in the expression profiles among the three cell types.