Systematic calibration of epitranscriptomic atlas using a synthetic modification-free RNA library
Recent years has witnessed rapid progress of the field epitranscriptomics. Functional interpretation of epitranscriptome relies on mapping technologies which determine the localization and stoichiometry of various RNA modifications. However, contradictory results are derived from different studies, questioning the biological impacts of certain RNA modifications. Here, we develop an approach for the generation of synthetic RNA library resembling the endogenous transcriptome but lacking modifications. Incorporating this modification-free RNA library as a negative control into established techniques, we obtain precise and quantitative maps of m6A and m5C after removing the pervasive false positives resulted from other elements such as specific sequence context and RNA secondary structure. Overall design: In this study, we developed a new method to create a synthetic RNA library resembling the sequence and expression of endogenous transcriptome except modifications. Then we applied the modification-free RNA library in three representative scenarios described above and obtained high confidence, quantitative epitranscriptome maps of m6A and m5C after eliminating the false positive sites.