Abstract
To study the direct effects of R-loops on DNA replication and other DNA-templated processes in vitro, R-loop-containing DNA templates need to be prepared efficiently and to near homogeneity. Here, we describe a simple transcription-based approach to form R-loops on plasmid DNA templates in vitro. We detail steps to transcribe a DNA sequence element with a high propensity to form co-transcriptional R-loops using T7 RNA polymerase. We describe nucleolytic digestion of free RNA, deproteinization, and repurification of R-loop-containing templates via gel filtration. For complete details on the use and execution of this protocol, please refer to Kumar et al.1.