The authors describe a new chromatin profiling method called Cleavage Under Targets and Release Using Nuclease (CUT&RUN), which uses antibody-targeted controlled cleavage by micrococcal nuclease to release specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), CUT&RUN is performed in situ, allowing for both 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, robust, and requires only about one-tenth the sequencing depth of ChIP, making it 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. The authors conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.The authors describe a new chromatin profiling method called Cleavage Under Targets and Release Using Nuclease (CUT&RUN), which uses antibody-targeted controlled cleavage by micrococcal nuclease to release specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), CUT&RUN is performed in situ, allowing for both 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, robust, and requires only about one-tenth the sequencing depth of ChIP, making it 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. The authors conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.