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In 30 consecutive analyses, the relative standard deviation (RSD) of the 5hmdC and 5fdC peak areas is 2.0% and 3.2%, respectively. In this case, no stable isotope-labeled standard is required for internal calibration. We further performed a comprehensive profiling of DNA cytosine modifications in 26 tissues of age-different C57BL/6N mice. Interestingly, we found that only liver 5hmdC abundance increases with the increasing age of adult mice, suggesting that liver 5hmdC might be a potential indicator of age in adulthood.Low-molecular-we