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The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine

Abstract

5-Hydroxymethylcytosine (5hmC) is a newly discovered DNA base present at detectable levels in most mammalian cell types and tissues. It is generated by Tet-enzyme–mediated oxidation of 5-methylcytosine (5mC). 5hmC is important both because of its potential role in regulating gene expression and because it may be an intermediate in DNA demethylation. Here we describe a technique termed GLIB (glucosylation, periodate oxidation and biotinylation), which combines several enzymatic and chemical modification steps to attach biotin to 5hmC. Biotin-containing genomic DNA fragments are then enriched using streptavidin beads, eluted and sequenced. GLIB is capable of quantitatively tagging and precipitating fragments containing a single 5hmC molecule. Sample preparation and GLIB can be conducted in 2–3 d.

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Figure 1: The GLIB method.
Figure 2: Effect of sonication on background precipitation in genomic DNA samples.
Figure 3
Figure 4
Figure 5: Anticipated results of experiments to verify successful glucosylation.

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Acknowledgements

W.A.P. was supported by a predoctoral graduate research fellowship from the National Science Foundation. Y.H. is supported by a postdoctoral fellowship from the Leukemia and Lymphoma Society. This study was supported by US National Institutes of Health (NIH) grants AI44432 and HD065812, grant RM1-01729 from the California Institute for Regenerative Medicine and Translational Research grant 6187-12 from the Leukemia and Lymphoma Society (to A.R.) as well as NIH grant HL089150 and a pilot grant from the Harvard Clinical and Translational Science Center (NIH grant 1 UL1 RR 025758-02) (to S.A.).

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A.R. and S.A. conceptualized and directed the project. W.A.P. and Y.H. developed the GLIB method. H.R.H. contributed to optimizing the GLIB method.

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Correspondence to Anjana Rao.

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The authors declare no competing financial interests.

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Pastor, W., Huang, Y., Henderson, H. et al. The GLIB technique for genome-wide mapping of 5-hydroxymethylcytosine. Nat Protoc 7, 1909–1917 (2012). https://doi.org/10.1038/nprot.2012.104

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