C&R
Product Information
Product Usage Information
For the pAG-MNase Enzyme, after cell permeabilization and primary antibody binding, resuspend cells in 50 μl of digitonin buffer containing 1.5 μl of pAG-MNase Enzyme (33X dilution). Incubate cell samples with rotation at 4°C for 1 hour, wash cells with digitonin buffer, and then perform the chromatin digestion.
Sample Normalization Spike-In DNA can be added directly to the digestion stop buffer. For sample normalization with NG-seq, add 5 μl (50 pg) of Sample Normalization Spike-In DNA
to each reaction. When using 100,000 cells or 1mg of tissue per reaction this ensures that the normalization reads are around 0.5% of the total sequencing reads. If more or less than 100,000 cells or 1mg of tissue are used per reaction, proportionally scale the volume of Sample Normalization Spike-In DNA up or down to adjust normalization reads to around 0.5% of total reads.When performing sample normalization, be sure to map the CUT&RUN sequencing data for all samples to both the test reference genome (e.g. human) and the sample normalization genome (S. cerevisiae).
Storage
Product Description
Background
Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets and 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). 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 signal between samples and between experiments.
Species Reactivity
Species reactivity is determined by testing in at least one approved application (e.g., western blot).
Applications Key
C&R: CUT&RUN
Cross-Reactivity Key
H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected
Trademarks and Patents
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