CUT&RUN is a chromatin profiling method that allows high-resolution mapping of DNA-binding protein sites. It uses antibody-targeted micrococcal nuclease to cleave and release specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes chromatin, CUT&RUN is performed in situ, enabling quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making it especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long-range contact sites at high resolution. CUT&RUN provides robust TF occupancy maps with improved sensitivity/specificity trade-offs relative to ChIP-seq. It can map large mobile chromatin complexes, even those that are rare, insoluble and AT-rich. CUT&RUN also maps histone modifications in compacted chromatin and directionally maps long-range genomic contacts. It allows quantitative measurements with low cell numbers and is widely applicable for chromatin profiling. CUT&RUN is an attractive alternative to ChIP-seq for high-resolution mapping of protein-DNA interactions.CUT&RUN is a chromatin profiling method that allows high-resolution mapping of DNA-binding protein sites. It uses antibody-targeted micrococcal nuclease to cleave and release specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes chromatin, CUT&RUN is performed in situ, enabling quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making it especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long-range contact sites at high resolution. CUT&RUN provides robust TF occupancy maps with improved sensitivity/specificity trade-offs relative to ChIP-seq. It can map large mobile chromatin complexes, even those that are rare, insoluble and AT-rich. CUT&RUN also maps histone modifications in compacted chromatin and directionally maps long-range genomic contacts. It allows quantitative measurements with low cell numbers and is widely applicable for chromatin profiling. CUT&RUN is an attractive alternative to ChIP-seq for high-resolution mapping of protein-DNA interactions.