CUT&RUN Assay Kit (with Drosophila Spike-In Control) #84647
- C&R
Product Information
Storage
Protocol
Product Description
The CUT&RUN Assay Kit (with Drosophila Spike-in Control) is conveniently designed to provide the reagents required to perform up to 24 CUT&RUN reactions, allowing for normalization of variabilities across the entire experiment. The kit is optimized for 5,000–250,000 cells per reaction and validated for a wide range of DNA-binding proteins, including histones, transcription factors, and cofactors. The kit has also been validated with fixed cells, primary cells, and tissues. A complete assay can be performed in as little as one day, and enriched DNA can be analyzed by downstream next-generation sequencing (NGS) and quantitative PCR (qPCR).
This kit includes a complete sample normalization strategy that starts with the addition of cryopreserved Drosophila spike-in nuclei to cell samples prior to performing the CUT&RUN assay. Like the cells of interest, the spike-in nuclei also bind to the Concanavalin A beads, providing a method to immobilize the spike-in nuclei for subsequent washes and incubations. During the antibody incubation step, a Drosophila H2Av antibody is added to the reaction in addition to the antibody against the target of interest. This Drosophila H2Av antibody provides a reliable method to target, digest, and enrich histone H2Av-bound Drosophila chromatin in a consistent way across all samples. This enrichment of H2Av-bound Drosophila chromatin occurs in the same tube as the enrichment of the target of interest from the test cell chromatin, ensuring that the spike-in control nuclei are subjected to all of the same steps and conditions as the test cells. A normalization factor is generated based on the Drosophila chromatin enrichment signal and is applied to the test genome to normalize signals across samples (see Figures 1 and 2). Thus, this spike-in normalization approach enables reliable and consistent normalization across samples, correcting for variations in starting cell number, technical variation among samples during processing, global histone modification changes, and non-specific enrichment coming from isotype control antibodies (i.e., non-specific rabbit IgG).
This kit provides three 165 µL vials of cryopreserved spike-in Drosophila nuclei. Each vial contains sufficient nuclei for eight CUT&RUN reactions using 100,000-250,000 cells or 1–2.5 mg of tissue, when used at the recommended volume. Also included is the H2Av Rabbit Monoclonal Antibody, which specifically detects the Drosophila histone variant H2Av, showing no cross-reactivity to mammalian histones, and a Drosophila Normalization Primer Set to facilitate sample normalization in downstream qPCR analysis.
In addition to the spike-in normalization reagents described above, the kit provides additional key experimental controls to measure the success of the CUT&RUN assay. These include the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751 and negative control Rabbit (DA1E) Monoclonal Antibody IgG Isotype Control (CUT&RUN) #66362, both of which can be used for downstream qPCR and NGS analysis. PCR primer sets are included for the human (#7014) and mouse (#7015) RPL30 loci for use in conjunction with the positive and negative control antibodies.
Specificity / Sensitivity
The CUT&RUN Assay Kit (with Drosophila Spike-in Control) can be used with any CUT&RUN-validated antibody to detect endogenous protein–DNA interactions and histone modifications in cells and tissues (see Figures 1-6). This kit is compatible with multiple antibody species, including rabbit and mouse. The positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751 detects tri-methyl-H3K4 across multiple species (human, mouse, rat, and monkey).
Because the H2Av Rabbit Monoclonal Antibody specifically detects only Drosophila chromatin, the normalization strategy can be applied broadly to experiments across multiple species without cross-reactivity. The included primer sets for human (#7014) and mouse (#7015) RPL30 loci serve as positive controls; for other species, additional control primer sets should be designed. The Drosophila Normalization Primer Set specifically recognizes Drosophila DNA for normalization purposes.
Background
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 cells or tissue to purified DNA, and has been shown to work with as few as 5,000-10,000 cells per assay (Figures 2-4). 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).
A major challenge in comparing CUT&RUN datasets is being able to account for differences in signal resulting from sample-to-sample variability, such as differences in starting cell number, technical variations among samples during processing, and variations in global histone modifications. The CUT&RUN Assay Kit (with Drosophila Spike-in Control) provides a complete normalization strategy that allows for normalization of variabilities across the entire experiment (5,6), ensuring a more accurate quantification of protein–DNA interactions between samples and across experiments.
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