CUT&RUN Assay Kit (#86652) Datasheet with Images Cell Signaling Technology

For Research Use Only. Not for Use in Diagnostic Procedures.

Product Includes	Mini Kit (P) Quantity	Standard Kit (S) Quantity	Storage Temp
Concanavalin A Magnetic Beads	1 x 80 µl	1 x 240 µl	+4°C
Concanavalin A Bead Activation Buffer	1 x 1.7 mL	1 x 5 mL	+4°C
Antibody Binding Buffer (CUT&RUN, CUT&Tag) 15338	1 x 800 µl	1 x 2.5 ml	+4°C
10X Wash Buffer (CUT&RUN, CUT&Tag) 31415	1 x 4.5 mL	1 x 15 ml	+4°C
CUT&RUN DNA Extraction Buffer 42015	1 x 3.6 mL	1 x 7 ml	+4°C
Calcium Chloride	1 x 25 µl	1 x 100 µl	+4°C
pAG-MNase Enzyme	1 x 20 µl	1 x 40 µl	-20°C
Digitonin Solution 16359	1 x 1.2 mL	2 x 1.2 ml	-20°C
CUT&RUN 4X Stop Buffer 48105	1 x 300 µl	1 x 1 ml	-20°C
100X Spermidine 27287	1 x 400 µl	1 x 1.3 ml	-20°C
Protease Inhibitor Cocktail (200X) 7012	1 x 200 µl	1 x 750 µl	-20°C
Proteinase K (20 mg/ml) 10012	1 x 20 µl	1 x 100 µl	-20°C
RNAse A (10 mg/ml) 7013	1 x 20 µl	1 x 50 µl	-20°C
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb 9751	1 x 20 µl	1 x 20 µl	-20°C
Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) 66362	1 x 20 µl	1 x 100 µl	-20°C
Sample Normalization Spike-In DNA (1 ng/μl)	1 x 40 µl	1 x 120 µl	-20°C
Sample Normalization Primer Set	1 x 64 µl	1 x 150 µl	-20°C
SimpleChIP® Human RPL30 Exon 3 Primers 7014	1 x 50 µl	1 x 150 µl	-20°C
SimpleChIP® Mouse RPL30 Intron 2 Primers 7015	1 x 50 µl	1 x 150 µl	-20°C

Applications:

C&R

Product Information

Storage

All components in this kit are stable for at least 12 months when stored at the recommended temperature.

Specificity / Sensitivity

The CUT&RUN Assay Kit can be utilized with any CUT&RUN-validated antibody to detect endogenous levels of protein-DNA interactions and histone modifications in mammalian cells (see Figures 1–6). The kit is compatible with multiple species of antibodies, including rabbit and mouse. The positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 detects multiple species of tri-methyl histone H3 Lys4 protein, including human, mouse, rat, and monkey. Primer sets are included for the human (#7014) and mouse (#7015) positive control RPL30 gene locus; however, the use of other species with the kit requires the design of additional control primer sets.

Product Description

The CUT&RUN Assay Kit is designed to conveniently provide reagents needed to perform up to 8 (P size) or 24 (S size) 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.
The CUT&RUN Assay Kit also provides important controls to ensure a successful CUT&RUN experiment. The kit contains a positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 and a negative control Rabbit (DA1E) mAb IgG XP^® Isotype Control (CUT&RUN) #66362, both of which can be used for qPCR or Next Generation sequencing (NG-seq) analysis. PCR primer sets are provided for the human (#7014) and mouse (#7015) RPL30 gene locus to be used in conjunction with the control antibodies. This kit is compatible with both qPCR and NG-seq.

Background

Like the chromatin immunoprecipitation (ChIP) assay, Cleavage Under Targets & 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). This assay can be used to identify multiple proteins associated with a specific region of the genome, or the opposite, to identify the many regions of the genome associated with a particular protein. In addition, the CUT&RUN assay can be used to define the spatial and temporal relationship of a particular protein-DNA interaction. For example, the CUT&RUN assay can be used to determine the specific order of recruitment of various protein factors to a gene promoter or to “measure” the relative amount of a particular histone modification across an entire gene locus during gene activation. In addition to histone proteins, the CUT&RUN assay can also be used to analyze binding of transcription factors and cofactors, DNA replication factors, and DNA repair proteins (Figures 1-6).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 500-1000 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 of 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

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.

SimpleChIP is a registered trademark of Cell Signaling Technology, Inc.

XP is a registered trademark of Cell Signaling Technology, Inc.

U.S. Patent No. 7,429,487.

All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

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