Description
Storage
All components in this kit are stable for at least 12 months when stored at the recommended temperature.
Protocol
Available protocols: CUT&RUN
Specificity / Sensitivity
The CUT&RUN Assay Kit can be utilized with any CUT&RUN-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells (see Figures 1-6). The kit is compatible with multiple species of antibodies, including rabbit and mouse. The positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751 detects multiple species of tri-methyl histone H3 Lys4 protein, including human, mouse, rat, and monkey. Primer sets are included for the human (#7014) and mouse (#7015) positive control RPL30 gene locus; however, the use of other species with the kit requires the design of additional control primer sets.
Background
Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a powerful and versatile technique used for probing protein-DNA interactions within the natural chromatin context of the cell (1-4). This assay can be used either to identify multiple proteins bound to a single, specific genomic region (by using different antibodies), or conversely, to map the various genomic regions associated with a single protein of interest. In addition, the CUT&RUN assay can be used to define the spatial and temporal relationship of a particular protein-DNA interaction. For example, the CUT&RUN assay can be used to determine the specific order of recruitment of various protein factors to a gene promoter or to "measure" the relative amount of a particular histone modification across an entire gene locus during gene activation. In addition to histone proteins, the CUT&RUN assay can also be used to analyze binding of transcription factors and cofactors, DNA replication factors, and DNA repair proteins (Figures 1-6). CUT&RUN provides a rapid, robust, and true low cell number assay for detection of protein-DNA interactions in the cell. Unlike the ChIP assay, CUT&RUN is free from formaldehyde cross-linking, chromatin fragmentation, and immunoprecipitation, making it a much faster and more efficient method for enriching protein-DNA interactions and identifying target genes. CUT&RUN can be performed in less than one day, from cells or tissue to purified DNA, and has been shown to work with as few as 5,000-10,000 cells per assay (Figures 1-4). Instead of fragmenting all of the cellular chromatin as done in ChIP, CUT&RUN utilizes an antibody-targeted digestion of chromatin, resulting in much lower background signal than seen in the ChIP assay. As a result, CUT&RUN requires only 1/10th of the sequencing depth that is required for ChIP-seq assays (1,2). Finally, the inclusion of yeast spike-in control DNA allows for accurate quantification and normalization, correcting for technical variations among samples during DNA purification, library preparation, and NGS steps.
Alternate Names
CnR; Cut & Run; CUT & Tag; cut and run; Cut and Run; cut and tag; CUT&RUN; CUT&Tag; cutandrun; cutandtag; histone modification
