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Abstract

RNA:DNA hybrids not only function in various physiological cellular processes but also represent a threat to genome integrity. The methyl-5-cytosine (m⁵C) marked RNA:DNA hybrids containing the m⁵C modified RNA strand add another layer of regulation to the R-loop structure. Here we describe the detection of m⁵C modifications in the context of RNA:DNA hybrids using immunostaining, and identification of specific binding partners of m⁵C marked RNA:DNA hybrids using pull-down method.