Cell Signaling Technology
Get fast and accurate results with CUT&RUN assay kits
By Maria Kam Eriksen, BioNordika Denmark
The Cleavage Under Targets & Release Using Nuclease (CUT&RUN) assay is a faster, more cost-effective alternative to ChIP-qPCR and ChIP-seq that requires fewer cells to investigate protein-DNA interactions. Cell Signaling Technology® (CST) has developed a CUT&RUN Assay kit designed to conveniently provide reagents needed to perform up to 24 digestion reactions from cells and is optimized for 100,000 cells per reaction.
The kit has been optimized to work for all types of DNA binding proteins, including histones, transcription factors and cofactors.
A complete assay can be performed in as little as one day. All you need to provide is a primary antibody against your protein of interest.CST provides everything else in the CUT&RUN kit (#86652) that contains all the buffers and reagents you need, along with a detailed protocol. The CUT&RUN Assay Kit also provides important controls to ensure a successful CUT&RUN experiment.
What is CUT&RUN?
The development of methods that enable mapping of protein-DNA interactions, such as chromatin-immunoprecipitation (ChIP) and ChIP-seq, have led to a growing awareness that aberrant epigenetic regulation drives a wide variety of human diseases. Cleavage Under Targets & Release Using Nuclease (CUT&RUN) is a new technology that can be used for chromatin profiling and a low cell number alternative to ChIP-qPCR and ChIP-seq.
Expanded benefits
The CUT&RUN Assay Kit now offers these additional benefits to increase assay flexibility
- Improved protocols: Optimized protocols enhance the enrichment of low abundance and/or weak binding transcription factors and cofactors
- Even lower sample requirement: 5-10K cells for Histones. 10-20K cells for Transcription Factors and Cofactors
- Now compatible with fixed cells: Light cell fixation keeps cells intact, preserves cell signaling pathways, and enhances the enrichment for accessory components of huge complexes
- Use fixed or fresh tissue samples with confidence: Validated and optimized tissue protocols give you data confidence when using ~20x less sample compared to ChIP
- Study protein-DNA interactions in primary cells: CUT&RUN significantly lowers the cell number requirements, making it suitable to use with primary cells
Plus, don’t forget that the CUT&RUN Assay Kit from CST provides
- Fast time to results: 1-2 days from cell to DNA
- Lower sequencing depth = lower sequencing costs: Only requires 3-5 million high-quality reads per sample due to the inherently low assay background
- In Vivo Method: Assays are performed using native chromatin, eliminating cross-linking artifacts
- Antibody versatility: Compatible with rabbit and mouse antibodies
- Target versatility: Generate sequencing and/or qPCR data for histones, his- tone modifications, transcription factors, and cofactors.
- Straightforward quantification: Spike in control DNA to simplify data quantification and normalization
How CUT&RUN works
CUT&RUN is an in vivo method that uses a target-specific primary antibody and a Protein A-Protein G-Micrococcal Nuclease (pAG-MNase) to isolate specific protein-DNA complexes1,2,3. It only takes 1 to 2 days to get from cell to DNA and can be automated for maximal throughput and reproducibility4.
To isolate the protein-DNA complex of interest, cells are first harvested and bound to Concanavalin A-coated magnetic beads to simplify cell handling and minimize cell loss during subsequent washes. Cell membranes are permeabilized with digitonin to facilitate the entry of the primary antibody into the nuclei, where it binds to the histone, transcription factor, or cofactor of interest. The pAG domain of the pAG-MNase fusion protein then binds to the primary antibody heavy chain, targeting the enzyme to the chromatin region of interest. The addition of Ca2+ activates the pAG-MNase to initiate DNA digestion. This allows the cleaved chromatin complex to diffuse away from the genomic chromatin, out of the nuclei, and into the sample supernatant, where it can be collected using either a DNA spin column or phenol/chloroform extraction followed by ethanol precipitation.
The purified, enriched DNA can then be identified and quantified using qPCR or used to construct a DNA sequencing library for next-generation sequencing (NGS) and whole-genome mapping.
Links
CUT&RUN Frequently Asked Questions
CUT&RUN Protocol
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