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. 2020 Aug;26(8):1038–1048. doi: 10.1261/rna.075275.120

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© 2020 Henry et al.; Published by Cold Spring Harbor Laboratory Press for the RNA Society

This article is distributed exclusively by the RNA Society for the first 12 months after the full-issue publication date (see http://rnajournal.cshlp.org/site/misc/terms.xhtml). After 12 months, it is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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FIGURE 1. — Detecting m⁵C modification on EBER1 by LC-MS/MS analysis. (A) Schematic outline of isolating native EBERs from total RNA followed by mass spec analysis. EBERs are isolated by ASO selection under denaturing conditions and eluted from the beads with TEACl buffer followed by denaturing PAGE. Please note that EBER1-RNPs are refractory to ASO-selection, as no regions are available for hybridization, while naked EBER1 RNA can be purified by ASOs. Size-selected EBERs are subjected to LC-MS/MS analysis. (B) SYBR Gold-stained polyacrylamide gel of purified native EBER1 and EBER2. Arrow indicates the EBERs. (C) Northern blot analysis of the gel-excised EBER1 and EBER2 to confirm correct purification. (D,E) LC-MS/MS trace for m⁵C modification. EBER1 contains approximately one m⁵C modification per RNA molecule, while EBER2 carries no chemical modification.