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bioRxiv · 10.1101/2021.03.23.434325

Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation

Fabyanic, E. B.·Hu, P.·Qiu, Q.·Berrios, K. N.·Flournoy, J.·Connolly, D. R.·Zhou, Z.·Kohli, R. M.·Wu, H.
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Abstract

Oxidative modification of 5-methylcytosine (5mC) generates 5-hydroxymethylcytosine (5hmC), a DNA modification that exhibits unique epigenetic regulatory functions and impacts diverse biological processes. However, standard single-nucleus/cell bisulfite sequencing methods cannot resolve the base ambiguity between 5mC and 5hmC to accurately measure cell-type specific epigenomic patterns and gene regulatory functions of 5hmC or true 5mC. Here, we develop single-nucleus 5hmC sequencing (snhmC-seq) for quantitative and unbiased 5hmC profiling in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. We used snhmC-seq to profile single nuclei from cryopreserved mouse brain samples to reveal epigenetic heterogeneity of 5hmC at single-cell resolution and uncovered a non-canonical gene regulatory role of genic 5hmC in non-CG context.

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Fabyanic, E. B., Hu, P., Qiu, Q., Berrios, K. N., Flournoy, J., Connolly, D. R., Zhou, Z., Kohli, R. M., Wu, H.. 2021-03-24. Quantitative single cell 5hmC sequencing reveals non-canonical gene regulation by non-CG hydroxymethylation. https://doi.org/10.1101/2021.03.23.434325

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