Total RNA was isolated using Trizol RNA extraction. Small RNAs were subsequently purified from total RNA using the Zymo RNA Clean & Concentrator-5 kit. Small RNAs (1 μg) was digested and analyzed by LC-MS as previously described (Dewe et al., 2017 ; Zhang et al., 2020 (link)) (Cai et al, 2015 (link)). Briefly, ribonucleosides were separated using a Hypersil GOLD C18 Selectivity Column (Thermo Scientific) followed by nucleoside analysis using a Q Exactive Plus Hybrid Quadrupole-Orbitrap. The modification difference ratio was calculated using the m/z intensity values of each modified nucleoside following normalization to the sum of intensity values for the canonical ribonucleosides; A, U, G and C. Statistical analysis of the mass spectrometry results was performed using GraphPad Prism software with error bars representing the standard deviation. Statistical tests and the number of times each experiment was repeated are noted in the figure legend. Raw intensity values for each measured nucleoside are provided in the source data file.
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Quantitative Analysis of RNA Modifications
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Other organizations : University of Rochester, Institut de Recherche en Infectiologie de Montpellier, Université de Montpellier, Centre National de la Recherche Scientifique
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Variable analysis
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- Ribonucleosides were separated using a Hypersil GOLD C18 Selectivity Column (Thermo Scientific)
- Nucleoside analysis using a Q Exactive Plus Hybrid Quadrupole-Orbitrap
- Modification difference ratio calculated using the m/z intensity values of each modified nucleoside following normalization to the sum of intensity values for the canonical ribonucleosides; A, U, G and C
- Total RNA was isolated using Trizol RNA extraction
- Small RNAs were subsequently purified from total RNA using the Zymo RNA Clean & Concentrator-5 kit
- Small RNAs (1 μg) was digested and analyzed by LC-MS as previously described (Dewe et al., 2017 (no link found); Zhang et al., 2020 (link)) (Cai et al, 2015 (link))
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