Learn about CUT&RUN from Cell Signaling Technology® (CST) – a low cell number alternative to ChIP-qPCR and ChIP-seq to analyze protein-DNA interaction in chromatin.
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 Target & Release Using Nuclease (CUT&RUN) is a new technology that can be used for chromatin profiling.
How It 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 complexes. It only takes 1 to 2 days to get from cell to DNA and can be automated for maximal throughput and reproducibility.
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.
Cell Signaling Technology® (CST) offers a CUT&RUN assay that overcomes many of the challenges faced with other whole-genome mapping techniques. Benefits offered by the CST CUT&RUN Assay Kit include:
|Low sample requirement
|Only 100K cells needed
|Fast time to results
|1 to 2 days from cell to DNA
|Sequencing cost savings
|Only 3 to 5 million high-quality reads required
|Generate sequencing and/or qPCR data for histones, histone modifications, transcription factors, and cofactors
|Compatible with rabbit and mouse antibodies
|Spike in control DNA to normalize signal between samples
|Avoid “crosslinking” artifacts
|An in vivo method performed using native chromatin
|CUT&RUN Assay Kit
|CUT&RUN pAG-MNase and Spike-in DNA
|DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN)
|SimpleChIP® Universal qPCR Master Mix
|SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina®
|SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Dual Index Primers)
|SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® (Single Index Primers)
|Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN)
|Concanavalin A Magnetic Beads and Activation Buffer
|Protease Inhibitor Cocktail (200X)
|RNAse A (10 mg/ml)
|Proteinase K (20mg/ml)
Do you need help choosing the right product for you? Please visit the CST website https://www.cellsignal.com/ or contact us at BioNordika.
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