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CUT&RUN pAG-MNase and Spike-In DNA

CUT&RUN pAG-MNase and Spike-In DNA #40366

Name: CUT&RUN pAG-MNase and Spike-In DNA
Price: 320.00 USD
Availability: InStock

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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

pAG-MNase Enzyme is supplied in 10 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1mM EDTA, 0.1mM PMSF and 50% glycerol. Do not aliquot the product. Sample Normalization Spike-In DNA is supplied in 10 mM Tris-HCl (pH 8.0). Store both products at –20°C. These products are stable for at least 12 months.

Protocol

Product Description

The CUT&RUN pAG-MNase and Spike-In DNA kit provides enough enzyme and spike-in DNA to support 50 CUT&RUN assays. The pAG-MNase Enzyme #57813 is a fusion of Protein A and Protein G to Micrococcal Nuclease, and is recombinantly produced in E. coli. The pAG-MNase is compatible with multiple species of antibodies, including both rabbit and mouse. The Sample Normalization Spike-In DNA (10 pg/µl) #29987 is fragmented genomic DNA from the yeast S. cerevisiae that can be added to a CUT&RUN reaction to facilitate normalization between samples during NG-seq analysis.

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.

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