Here we describe a bench top protocol for Cleavage Under Targets and Tagmentation (CUT&Tag), an enzyme-tethering strategy that provides efficient high-resolution sequencing libraries for profiling diverse chromatin components. In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a proteinA-Tn5 transposase fusion protein. Activation of the transposase efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in one day. We have demonstrated the utility of CUT&Tag by profiling histone modifications, RNA Polymerase II and transcription factors on low cell numbers and single cells. Version 3 includes an optional light formaldehyde treatment of cells or nuclei prior to antibody addition. Light fixation reduces the tendency of cells or nuclei to clump, which can also be reduced for fixed and unfixed nuclei by omitting digitonin from all steps.