PROJECT SUMMARY/ABSTRACT DNA-binding proteins play crucial roles in all DNA templated processes, such as transcription, splicing, replication, and DNA repair. DNA binding proteins include transcription factors that bind preferentially to certain DNA sequences, and histone proteins that form the core of nucleosomes. Importantly, genomic location of factors or histone proteins cannot be predicted in cell types by DNA sequence alone. Therefore, protein profiling technologies are used to identify cell specific characteristics of functional binding. The importance of DNA-binding proteins has motivated the continued development of experimental and analytical methods to better identify and characterize these interactions. Genome-wide profiling by ChIP-seq is a widely-used technique that has assisted in the characterization of countless chromatin binding proteins. However, this technique is limited in its ability to characterize factor occupancy in samples with small cell numbers and by the availability of specific and robust antibodies. These limitations have necessitated the development of complementary methods and extensions of ChIP-seq to provide a more complete of biological processes in the cell. Very recently, we optimized CUT&RUN, a new localization method, to profile factor occupancy in extremely low cell populations, down to single cells and individual mouse blastocyst embryos (termed uliCUT&RUN). This technical advancement has opened the opportunity to profile factor occupancy in rare cell populations, such as patient biopsies. Furthermore, it permits for testing cell heterogeneity that occurs in cell populations. However, practical limitations of this technology still include antibody development and efficiency. Camelid single-chain VHH antibodies or Nanobodies (Nbs) are a compelling new class of antibodies characterized by exceptionally high solubility and thermostability. We have recently developed a robust pipeline for the discovery and characterization of high-quality antigen-specific Nb repertoires. This pipeline has been extensively tested and optimized for a dozen of antigens with different structures and immune responses. With this approach, a large cohort of high-quality conformational Nb binders can be identified. Here we propose to couple our expertise on Nb development and uliCUT&RUN to develop nanobody specific CUT&RUN for low cell populations and apply this technology to single cells and rare cell populations. The development and application of Nb-based uliCUT&RUN will be of wide use to the community and we are well poised to develop this technology given our optimization of CUT&RUN is the first time single cell transcription factor profiling has been accomplished and our expertise in the new field of Nb development. Further, results from applying this technology to samples will continue to further our understanding of normal cell biology, but also provide crucial information that will benefit efforts to determine the causes...