Protocol to prepare doubly labeled fluorescent nucleosomes for single-molecule fluorescence microscopy

Single-molecule fluorescence microscopy (SMFM) has been shown to be informa-tive in understanding the interaction of chromatin-associated factors with nucle-osomes, the basic building unit of chromatin. Here, we present a protocol for pre-paring doubly labeled fluorescent nucleosomes for SMFM. We describe steps for over-expression in E. coli and purification of recombinant human core histones. We then detail fluorescent labeling of histones and nucleosomal double -stranded DNA followed by octamer refolding and nucleosome reconstitution.