Base-resolution profiling of active DNA demethylation using MAB-seq and caMAB-seq
Autor: | Hao Wu, Xiaoji Wu, Yi Zhang |
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Rok vydání: | 2016 |
Předmět: |
0301 basic medicine
Sequence analysis genetic processes Bisulfite sequencing Computational biology Biology Molecular biology General Biochemistry Genetics and Molecular Biology DNA sequencing Sequencing by ligation ChIP-sequencing 03 medical and health sciences 030104 developmental biology DNA demethylation natural sciences Chromatin immunoprecipitation Epigenomics |
Zdroj: | Nature Protocols. 11:1081-1100 |
ISSN: | 1750-2799 1754-2189 |
DOI: | 10.1038/nprot.2016.069 |
Popis: | A complete understanding of the function of the ten-eleven translocation (TET) family of dioxygenase-mediated DNA demethylation requires new methods to quantitatively map oxidized 5-methylcytosine (5mC) bases at high resolution. We have recently developed a methylase-assisted bisulfite sequencing (MAB-seq) method that allows base-resolution mapping of 5-formylcytosine (5fC) and 5-carboxylcytosine (5caC), two oxidized 5mC bases indicative of active DNA demethylation events. In standard bisulfite sequencing (BS-seq), unmodified C, 5fC and 5caC are read as thymine; thus 5fC and 5caC cannot be distinguished from C. In MAB-seq, unmodified C is enzymatically converted to 5mC, allowing direct mapping of rare modifications such as 5fC and 5caC. By combining MAB-seq with chemical reduction of 5fC to 5hmC, we also developed caMAB-seq, a method for direct 5caC mapping. Compared with subtraction-based mapping methods, MAB-seq and caMAB-seq require less sequencing effort and enable robust statistical calling of 5fC and/or 5caC. MAB-seq and caMAB-seq can be adapted to map 5fC/5caC at the whole-genome scale (WG-MAB-seq), within specific genomic regions enriched for enhancer-marking histone modifications (chromatin immunoprecipitation (ChIP)-MAB-seq), or at CpG-rich sequences (reduced-representation (RR)-MAB-seq) such as gene promoters. The full protocol, including DNA preparation, enzymatic treatment, library preparation and sequencing, can be completed within 6-8 d. |
Databáze: | OpenAIRE |
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