Abstract
RNA transcripts can anneal with template DNA strands to form RNA-DNA hybrids (or R-loops if the non-template DNA strands exist), which play a variety of roles in many physiological processes. Here, we provide an accessible and reproducible approach for immunofluorescent staining of RNA-DNA hybrids with the S9.6 antibody in spread meiotic nuclei of Saccharomyces cerevisiae. This protocol allows the examination of RNA-DNA hybrids as clearly distinguishable foci and the colocalizations of RNA-DNA hybrids with other proteins. For complete details on the use and execution of this protocol, please refer to Yang et al. (2021).