Description
Product Usage Information
For the CUT&RUN or CUT&Tag assay, we recommend adding 1 μl 100X Spermidine # 27287 , 0.5 μl Protease Inhibitor Cocktail (200X) # 7012 , and 2.5 μl Digitonin Solution # 16359 to 96 μl Antibody Binding Buffer (CUT&RUN, CUT&Tag) (100 μl per reaction) right before use. Please keep on ice during use. The volume of Digitonin Solution # 16359 in the Antibody Binding Buffer (CUT&RUN, CUT&Tag) can be adjusted based on the specific cell line.
Storage
Store Antibody Binding Buffer (CUT&RUN, CUT&Tag) at 4°C. This product is stable for at least 12 months.
Background
Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets and Release Using Nuclease (CUT&RUN) and Cleavage Under Targets and Tagmentation (CUT&Tag) are powerful and versatile techniques used for probing protein-DNA interactions within the natural chromatin context of the cell (1-7). 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 live cells to purified DNA, and has been shown to work with as few as 500-1,000 cells per assay (1,2). 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 the sequencing depth that is required for ChIP-seq assays (1,2). Finally, the inclusion of simple spike-in control DNA allows for accurate quantification and normalization of target-protein binding that is not possible with the ChIP method. This provides for effective normalization of signals between samples and between experiments. CUT&Tag has many of the same advantages as the CUT&RUN assay in that it provides a rapid, robust, and true low cell number protocol for detection of protein-DNA interactions in the cell. In addition, the CUT&Tag assay adds an adaptor DNA ligation step carried out by the pAG-Tn5 enzyme, in which an adaptor DNA is ligated directly to antibody-targeted chromatin DNA fragments in the cell. As a result, subsequent DNA library preparation is much faster and easier than library preparation following the CUT&RUN assay, free from DNA end repair, A-tailing, and adaptor ligation . CUT&Tag works very well for analyzing histone modifications, in addition to mapping some transcription factors and cofactors binding.
Alternate Names
C&R; CnR; CUT & RUN; CUT & Tag; CUT & TAG; cut and run; CUT and RUN; CUT&RUN; CUT&Tag; cutandrun; CUTandRUN; CUTandTag; histone modification
