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.