The study introduces a method called Tet-assisted Bisulfite Sequencing (TAB-Seq) to map 5-hydroxymethylcytosine (5hmC) at single-base resolution across the mammalian genome. This method enables the quantification of both 5hmC and 5mC at each cytosine. By using β-glucosyltransferase to protect 5hmC from further oxidation and recombinant Tet1 to convert 5mC to 5caC, TAB-Seq distinguishes 5hmC from unmodified cytosine and 5mC. The method was applied to embryonic stem cells (ESCs), revealing widespread distribution of 5hmC, sequence bias, and strand asymmetry at 5hmC sites. 5hmC is enriched in promoter-distal regulatory elements and shows reciprocal low levels of 5mC near transcription factor binding sites. The base-resolution maps of 5hmC provide more accurate estimates of 5hmC and 5mC levels than traditional bisulfite sequencing. The study also shows that 5hmC is more abundant in distal regulatory elements than in gene bodies, and that 5hmC is strand-biased towards G-rich sequences. Additionally, 5hmC is most enriched near low CpG regions, suggesting a role in active demethylation. The results highlight the importance of 5hmC in epigenetic regulation and suggest that different mechanisms of DNA modification may be involved at distinct functional sequences in the genome. The study provides a robust method for analyzing 5hmC and 5mC at single-base resolution, which has implications for understanding the epigenetic landscape and developmental potential of cells.The study introduces a method called Tet-assisted Bisulfite Sequencing (TAB-Seq) to map 5-hydroxymethylcytosine (5hmC) at single-base resolution across the mammalian genome. This method enables the quantification of both 5hmC and 5mC at each cytosine. By using β-glucosyltransferase to protect 5hmC from further oxidation and recombinant Tet1 to convert 5mC to 5caC, TAB-Seq distinguishes 5hmC from unmodified cytosine and 5mC. The method was applied to embryonic stem cells (ESCs), revealing widespread distribution of 5hmC, sequence bias, and strand asymmetry at 5hmC sites. 5hmC is enriched in promoter-distal regulatory elements and shows reciprocal low levels of 5mC near transcription factor binding sites. The base-resolution maps of 5hmC provide more accurate estimates of 5hmC and 5mC levels than traditional bisulfite sequencing. The study also shows that 5hmC is more abundant in distal regulatory elements than in gene bodies, and that 5hmC is strand-biased towards G-rich sequences. Additionally, 5hmC is most enriched near low CpG regions, suggesting a role in active demethylation. The results highlight the importance of 5hmC in epigenetic regulation and suggest that different mechanisms of DNA modification may be involved at distinct functional sequences in the genome. The study provides a robust method for analyzing 5hmC and 5mC at single-base resolution, which has implications for understanding the epigenetic landscape and developmental potential of cells.