DNA tightropes are a powerful single-molecule microscopy platform for the study of protein interactions with DNA on a single-molecule level. DNA molecules are suspended between two beads, granting full 3D access for a protein to bind and its interactions to be studied. Furthermore, tightropes can be custom-designed, permitting the study of protein interactions that are sequence- or substrate-dependent such as base mismatches or lesions. In this chapter, we describe the methodology required to generate novel nucleosome tightropes from plasmid to flow chamber. We hope this technique will enable researchers to study DNA-binding protein interactions with chromatinized DNA shedding more light on the intricate processes happening within the context of chromatin.