Description
Product Usage Information
For the CUT&RUN and CUT&Tag assay, we recommend adding 25 μl of Digitonin Solution per 1 ml buffer. Not all cell lines exhibit the same sensitivity to digitonin, so it may be necessary to adjust the final volume of digitonin in the buffer to get optimal permeabilization of your specific cell line. Cell permeabilization can be tested by combining an equal volume of cell suspension and 0.4% Trypan Blue Stain. Optimal cell permeabilization will result in staining of >90% of the cell population.
Storage
Digitonin Solution is supplied in nuclease-free water. Store at -20°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
