To ensure lifelong persistence, all three human ?-herpesviruses establish latency in post-mitotic neurons. However, when neurons are exposed to physiological and genotoxic stresses, latent virus will restart the productive replication cycle (reactivate), producing new infectious virus that can spread into neighboring cells and be transmitted to new hosts. Repeated cycles of reactivation elicit a host inflammatory response that gives rise to mild to severe clinical disease. Available anti-viral drugs limit productive replication but there no licensed therapies that target latency or prevent reactivation and there are no fully effective vaccines. Prior studies using chromatin immunoprecipitation (ChIP) have concluded that latent viral genomes are assembled into heterochromatin that is refractive to transcription, thereby limiting the expression of viral genes. Because infected neurons represent a minority of cells in a ganglion, attempts to profile the epigenome of latent ?-herpesviruses using ChIP-seq have been unsuccessful. As a consequence, the molecular composition and distribution of heterochromatic factors associated with viral promoters and other regulatory sequences is only partially known. Studies in cultured neurons, where the proportion of infected neurons is much greater and more accessible to experimental manipulation, have shown that reactivation involves a stepwise reversal of epigenetic suppression permitting the de novo synthesis of viral effector proteins that orchestrate the transition into fully active euchromatin and initiation of viral DNA synthesis. Within this contextual framework, efforts by a number of laboratories including our own to elucidate the details of these steps has been held back by our inability to measure the distribution of specific proteins and protein modifications across the viral genome at high resolution using the relatively small numbers of latently infected neurons that can be obtained from neuron culture models. This represents a major roadblock to further progress by the field. To find a remedy, we plan to evaluate and if necessary, adapt, a new profiling technique called CUT&RUN, in which unfixed permeabilized cells are incubated with antibodies to specific chromatin-associated proteins or protein-modifications. After finding its target, the antibody recruits a protein A-micrococcal nuclease (pA/MNase) fusion protein that can be regulated by adding or removing calcium. The bound target/antibody/nuclease complex is excised by the nuclease and released into the supernatant where the DNA can be captured and identified by next-generation sequencing. This rapid technique offers high precision, high reproducibility, and does not require cross-linking or chromatin isolation. Importantly, the methodology can be applied to as few as 500 cells, which corresponds to a single well from the existing cultured neuron latency/reactivation models. Once we have optimized and validated the technique using different targets, it will likely become a routine method in studies of ?-herpesvirus latency.