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Precise genomic mapping of 5-hydroxymethylcytosine via covalent tether-directed sequencing

ABSTRACT: 5-hydroxymethylcytosine (5hmC) is the most prevalent intermediate on the oxidative DNA demethylation pathway and is implicated in regulation of embryogenesis, neurological processes and cancerogenesis. Profiling of this relatively scarce genomic modification in clinical samples requires cost-effective high resolution techniques that avoid harsh chemical treatment. Here, we present a bisulfite-free approach for 5hmC profiling at single nucleotide resolution, named hmTOP-seq, which is based on direct sequence readout primed at covalently labeled 5hmC sites from an in situ tethered DNA oligonucleotide. Examination of distinct tethering chemistries suggested a putative model for the targeted non-homologous polymerase priming which permits theoretical evaluation of suitable tethers at the design stage. The robustness and resolution of hmTOP-seq was validated on a small model genome and mouse embryonic stem cells, which allowed construction of single nucleotide 5hmCG maps that reflect subtle differences in strand-specific CG hydroxymethylation. Collectively, hmTOP-seq expands the toolbox of valuable techniques for cost-effective identification of epigenetic changes and new disease biomarkers.

ORGANISM(S): Mus musculus

PROVIDER: GSE140206 | GEO | 2020/04/17

REPOSITORIES: GEO

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