Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets & Release Under Nuclease (CUT&RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&RUN (uliCUT&RUN). Here, a detailed protocol is presented for single-cell factor profiling using uIiCUT&RUN in a manual 96-well format.

Automated summary

Determining the binding locations of a protein on chromatin is crucial for understanding its function. Chromatin Immunoprecipitation (ChIP) has been the gold standard for over 30 years, but CUT&RUN, a more sensitive antibody tethering technique, is gaining popularity. CUT&RUN is a powerful method for determining protein binding profiles, especially in low cell populations and single cells.