Product # | Size | Price |
---|---|---|
86652S | 1 Kit (24 assays) | $ 534 |
Product Includes | Volume (with Count) | Storage Temp | |||
---|---|---|---|---|---|
Concanavalin A Magnetic Beads | 1 x 240 µl | 4°C | |||
Concanavalin A Bead Activation Buffer | 1 x 5 ml | 4°C | |||
CUT&RUN Antibody Binding Buffer | 1 x 2.5 ml | 4°C | |||
CUT&RUN 10X Wash Buffer | 1 x 15 ml | 4°C | |||
CUT&RUN DNA Extraction Buffer | 1 x 7 ml | 4°C | |||
Calcium Chloride | 1 x 100 µl | 4°C | |||
pAG-MNase Enzyme | 1 x 40 µl | -20°C | |||
Digitonin Solution 16359 | 2 x 1.2 ml | -20°C | |||
CUT&RUN 4X Stop Buffer | 1 x 1 ml | -20°C | |||
100X Spermidine | 1 x 1.3 ml | -20°C | |||
Protease Inhibitor Cocktail (200X) 7012 | 1 x 750 µl | -20°C | |||
Proteinase K (20 mg/ml) 10012 | 1 x 100 µl | -20°C | |||
RNAse A (10 mg/ml) 7013 | 1 x 50 µl | -20°C | |||
Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb 9751 | 1 x 20 µl | -20°C | |||
Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) 66362 | 1 x 100 µl | -20°C | |||
Sample Normalization Spike-In DNA (1 ng/μl) | 1 x 120 µl | -20°C | |||
Sample Normalization Primer Set | 1 x 150 µl | -20°C | |||
SimpleChIP® Human RPL30 Exon 3 Primers 7014 | 1 x 150 µl | -20°C | |||
SimpleChIP® Mouse RPL30 Intron 2 Primers 7015 | 1 x 150 µl | -20°C |
Product Information
All components in this kit are stable for at least 6 months when stored at the recommended temperature.
! | This ! signifies an important step in the protocol regarding volume changes based on the number of CUT & RUN reactions being performed. |
!! | This !! signifies an important step to dilute a buffer before proceeding. |
SAFE STOP | This is a safe stopping point in the protocol, if stopping is necessary. |
NOTE: Steps for cell preparation (step 6-16) should be performed in succession at room temperature to minimize stress on the cells. To minimize DNA fragmentation, avoid vigorous vortexing and cavitation during resuspension.
NOTE: This protocol is written for the use of 100,000 cells per reaction. However, these same reaction conditions can be used for 10,000 to 250,000 cells per sample.
NOTE: The amount of digitonin recommended for cell permeabilization is in excess and should be sufficient for permeabilization of most cell lines. However, not all cell lines exhibit the same sensitivity to digitonin. Before you begin your experiment, it is recommended that you test your specific cell line by following the protocol provided in Appendix A. Digitonin treatment should result in permeabilization of >90% of the cell population.
! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
NOTE: Avoid vortexing of the Concanavalin A Magnetic Bead suspension as repeated vortexing may displace the Concanavalin A from the beads.
NOTE: To avoid loss of beads, remove liquid using a pipetman. Do not aspirate using a vacuum.
NOTE: The input sample will be incubated at 55°C later in the protocol, so it is recommended to use a safe-lock 1.5 ml tube to reduce evaporation during the incubation.
NOTE: Concanavalin A Magnetic Beads may clump or stick to the sides of the tube. Beads can be resuspended by pipetting up and down.
NOTE: The amount of antibody required for CUT&RUN varies and should be determined by the user. For the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb, add 2 µl of antibody to the sample. For the negative control Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362, add 5 µl to the sample. We strongly recommend using the negative control antibody and NOT a no-antibody control, because the latter results in high levels of non-specific MNase digestion and high background signal. We recommend using the input sample for comparison with both qPCR and NG-seq analysis.
! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
! All buffer volumes should be increased proportionally based on the number of CUT&RUN reactions being performed.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
NOTE: Digestion should be performed in a 4°C cooling block or refrigerator. The temperature of ice can get as low as 0°C, which can limit digestion and decrease signal.
NOTE: This incubation step can be increased to 30 min.
! All buffer volumes should be increased proportionally based on the number of input samples being prepared.
NOTE: Sonication conditions may need to be determined empirically by testing different sonicator power settings and/or durations of sonication, following the protocol in Appendix B. Optimal sonication conditions will generate chromatin fragments ranging in size from 100-600 bp. Sonication for 5 sets of 15-sec pulses using a VirTis Virsonic 100 Ultrasonic Homogenizer/Sonicator at setting 6 with a 1/8-inch probe sufficiently fragments the input chromatin.
DNA can be purified from input and enriched chromatin samples using DNA spin columns, as described in Section A, or phenol/chloroform extraction followed by ethanol precipitation as described in Section B. Purification using DNA spin columns is simple and fast, providing good recovery of DNA fragments above 35 bp (Figure 7A, Lane 2). Phenol/chloroform extraction followed by ethanol precipitation is more difficult, but provides better recovery of DNA fragments below 35 bp (Figure 7A, Lane 3); however, as shown in Figure 7B, the majority of DNA fragments generated in the CUT&RUN assay are larger than 35 bp. Therefore, DNA spin columns provide a quick and simple method for purification of > 98% of the total CUT&RUN DNA fragments.
Purified DNA can be quantified prior to NG-seq analysis using a picogreen-based DNA quantification assay. For CUT&RUN reactions containing 100,000 cells, the expected DNA yield for a CUT&RUN reaction ranges from 0.5 to 10 ng per reaction for transcription factors and cofactors, and 1 to 20 ng per reaction for histone modifications.
FIGURE 7. Comparison of DNA purification using spin columns or phenol/chloroform extraction followed by ethanol precipitation. (A) A low range DNA ladder mix (lane 1, unpurified) was purified using either DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (lane 2) or phenol/chloroform extraction followed by ethanol precipitation (lane 3) and separated by electrophoresis on a 4% agarose gel. As shown, phenol/chloroform followed by ethanol precipitation efficiently recovers all DNA fragment sizes, while DNA spin columns recover DNA fragments ≥ 35 bp. (B) DNA was purified using phenol/chloroform extraction followed by ethanol precipitation from a CUT&RUN assay performed using TCF4/TCF7L2 (C48H11) Rabbit mAb #2569. The size of the DNA fragments in the library was analyzed using a Bioanalyzer (Agilent Technologies). The adaptor and barcode sequences added to the library during construction account for 140 bp in fragment length. Therefore, starting 35 bp DNA fragments would be 175 bp in length after library preparation (indicated with blue vertical line in figure). As shown, less than 2% of the total CUT&RUN enriched DNA fragments are less than 175 bp (starting length of 35 bp), suggesting that DNA purification spin columns are sufficient for capture of > 98% of the total CUT&RUN DNA fragments.
NOTE: DNA can be purified from input and enriched chromatin samples using the Cell Signaling® DNA Purification Buffers and Spin Columns (ChIP, CUT&RUN) #14209 (not included in this kit) and the modified protocol below. Steps 1 through 5 are modified to reflect the requirement for adding 5 volumes (1.5 ml) of DNA Binding Buffer to the 300 µl of input and enriched chromatin samples.
NOTE: 5 volumes of DNA Binding Buffer should be used for every 1 volume of sample.
NOTE: The following reagents are required for the phenol/chloroform extraction and ethanol precipitation and are not included in this kit: phenol/chloroform/isoamyl alcohol (25:24:1), chloroform/isoamyl alcohol (24:1), 3M Sodium Acetate (pH 5.2), 20mg/ml glycogen, 100% ethanol, 70% ethanol, and 1X TE buffer or nuclease-free water.
NOTE: If sample normalization is performed, only the CUT&RUN samples are to be analyzed using the Sample Normalization Primer Set. The input DNA does not contain the Normalization Spike-In DNA.
Reagent | Volume for 1 PCR Reaction (18 µl) |
---|---|
Nuclease-free H2O #12931 | 6 µl |
5 µM Primers | 2 µl |
SimpleChIP® Universal qPCR Master Mix #88989 | 10 µl |
a. | Initial Denaturation | 95°C for 3 min |
b. | Denature | 95°C for 15 sec |
c. | Anneal and Extension | 60°C for 60 sec |
d. | Repeat steps b and c for a total of 40 cycles. |
C[T] value of Sample Normalization Primer Set | **Normalization Factor for qPCR | Signal Before Normalization (% Input Calc'd from Step 5) | Signal After Normalization | |
Sample 1 | 23.31 | 2(23.31-23.31)=1.00 | 24.4% | 24.4%/1.00=24.4% |
Sample 2 | 24.24 | 2(23.31-24.24)=0.52 | 12.0% | 12.0%/0.52=23.1% |
Sample 3 | 25.08 | 2(23.31-25.08)=0.29 | 6.28% | 6.28%/0.29=21.7% |
Sample 4 | 26.30 | 2(23.31-26.30)=0.13 | 2.72% | 2.72%/0.13=20.9% |
**Normalization Factor for qPCR = 2(C[T] Selected Sample - C[T] the Other Sample)
FIGURE 8. Normalization of CUT&RUN signals using spike in DNA for qPCR analysis. CUT&RUN was performed with a decreasing number of HCT116 cells and either Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb #9751 (upper panels) or Phospho-Rpb1 CTD (Ser2) (E1Z3G) Rabbit mAb #13499 (lower panels). Enriched DNA was quantified by real-time PCR using SimpleChIP® Human GAPDH Exon 1 Primers #5516, SimpleChIP® Human β-Actin Promoter Primers #13653, SimpleChIP® Human Β-Actin 3' UTR Primers #13669, and SimpleChIP® Human MyoD1 Exon 1 Primers #4490. The amount of immunoprecipitated DNA in each sample is represented as signal relative to the total amount of input chromatin for 100,000 cells. Non-normalized enrichments are depicted in the left panels. The Sample Normalization Spike-In DNA was added into each reaction proportionally to the starting cell number. Based on the difference of qPCR signals from spike in DNA in each sample, CUT&RUN signals were normalized to the sample containing 100,000 cells. Normalized enrichments are depicted in the right panels.
The immuno-enriched DNA samples prepared with this kit are directly compatible with NG-seq. For downstream NG-seq DNA library construction, use a DNA library preparation protocol or kit compatible with your downstream sequencing platform. For sequencing on Illumina® platforms, we recommend using the SimpleChIP® ChIP-seq DNA Library Prep Kit for Illumina® #56795 with SimpleChIP® ChIP-seq Multiplex Oligos for Illumina® #29580 or #47538.
The Number of Unique Reads Aligned to Yeast | Normalization Factor for NGS | The Number of Unique Reads Aligned to Test Reference Genome Before Normalization | The Number of Unique Reads Aligned to Test Reference Genome After Normalization | |
Sample 1 | 219,275 | 219,275/219,275 = 1.00 | 5,077,747 | 5,077,747 X 1.00 = 5,077,747 |
Sample 2 | 411,915 | 219,275/411,915 = 0.53 | 9,896,671 | 9,896,671 X 0.53 = 5,268,306 |
Sample 3 | 816,235 | 219,275/816,235 = 0.27 | 17,842,773 | 17,842,773 X 0.27 = 4,793,320 |
Sample 4 | 1,120,826 | 219,275/1,120,826 = 0.20 | 23,836,679 | 23,836,679 X 0.20 = 4,663,339 |
Normalization Factor for NGS = the number of unique yeast reads from Selected Sample / the number of unique yeast reads from the other sample
In the CUT&RUN protocol, the addition of digitonin to the buffers facilitates the permeabilization of cell membranes and entry of the primary antibody and pAG-MNase enzyme into the cells and nuclei. Therefore, having an adequate amount of digitonin in the buffers is critical to the success of antibody and enzyme binding and digestion of targeted genomic loci. Different cell lines show differing sensitivities to digitonin cell permeabilization. While the amount of digitonin recommended in this protocol should be sufficient for permeabilization of most cell lines, we recommend an initial test of your specific cell line. We have found that the addition of excess digitonin is not deleterious to the assay, so there is no need to perform a concentration curve. Rather, a quick test to determine if the recommended amount of digitonin works for your cell line is sufficient.
NOTE: Digitonin Solution should be stored at -20°C. Please keep on ice during use and store at -20°C when finished for the day.
Sonication of the input DNA sample is recommended because only fragmented genomic DNA (<10 kb) can be purified using DNA purification spin columns. Additionally, the fragmented genomic DNA (<1kb) may be used as the negative control in NG-seq analysis. Sonication should be optimized so that the input DNA is 100-600 bp in length.
We recommend using the input sample for NG-seq because it provides a convenient and unbiased representation of the cell genome. While the IgG sample can also be used as a negative control for NG-seq, it may show enrichment of specific regions of the genome due to non-specific binding. Unfragmented input DNA can be used for qPCR analysis. However, unfragmented DNA must be purified using phenol/chloroform extraction followed by ethanol precipitation.
! All buffer volumes should be increased proportionally based on the number of input samples being prepared.
NOTE: Samples will be incubated at 55°C in Step 9, so it is recommended to use a safe-lock 1.5 ml tube to reduce evaporation during the incubation.
Problem | Possible Causes | Recommendation |
---|---|---|
1. Concanavalin A beads clump during the experiment. | Bead clumping is normal and is not usually deleterious to the assay. | Resuspend clumped beads by gently pipetting up and down. |
Room temperature incubation of beads and cells is too long. | Activate Concanavalin A beads at 4°C and incubate with cells no longer than 5 min (Section I, Step 14). | |
Cells are lysing during preparation. | Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress (Section I, Steps 7-16). | |
Digitonin concentration may be too high. | Some cells may be more sensitive to digitonin and lyse at higher concentrations. Reduce the amount of digitonin in the assay, but be sure to confirm the amount used is sufficient for cell permeabilization (see APPENDIX A). | |
2. No DNA is detected in the purified DNA samples using a picogreen-based DNA quantification assay. | This is typical when starting with low cell numbers (<10,000 cells), but DNA should be detectable when starting with the recommended 100,000 cells. | Be sure to use a picogreen-based DNA quantification assay. Purified DNA is not typically detectable using a NanoDrop, Bioanalyzer® or Tapestation®. |
Cell count is off, cells are lost or lysing during preparation. | Starting cell culture should be 60-90% confluent and look healthy (> 90% live cells). | |
Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress. | ||
Wash all cells in one vial to minimize cell loss (Section I, Steps 7-16). | ||
Digitonin is not effectively permeabilizing the cells. | Be sure to store Digitonin Solution at -20°C when not in use, as it is unstable when stored above -20°C. | |
Be sure to test and confirm that the amount of digitonin used is sufficient to permeabilize your specific cell line (see APPENDIX A). | ||
pAG-MNase enzyme is not working properly in the assay. | The pAG-MNase is highly stable and should maintain activity for a long time when stored properly. | |
The pAG-MNase requires Ca2+ divalent cations for activity. Be sure to add calcium chloride for activation of the enzyme (Section III, Step 8). | ||
Be sure to digest for 30 min to allow the enzyme to sufficiently digest the chromatin (Section III, Step 9). | ||
Not enough antibody is added to the reaction or antibody does not work in the CUT&RUN assay. | Not all antibodies work in CUT&RUN. If possible, use a CUT&RUN validated antibody. Alternatively, some ChIP- and IF-validated antibodies also work for CUT&RUN. | |
Be sure to include the positive control Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit mAb to show your assay is working. | ||
3. No signal in qPCR or NG-seq analysis. | See possible causes for problem #2. | See recommendations problem #2. |
Not enough DNA added to the qPCR reaction. | Add more DNA to the PCR reaction or increase the number of amplification cycles. | |
Not enough DNA added to the NG-seq DNA library preparation. | Be sure to quantify the purified DNA using a picogreen-based DNA quantification assay and use the recommended amount of starting DNA and PCR-amplification cycles (see Section VII). | |
PCR-amplified region may span a nucleosome-free region. | DNA fragments generated in the CUT&RUN assay are typically smaller than DNA fragments generated in the ChIP assay. Therefore, it is critical to design primers to generate amplicons no longer than 60 to 80 bp. | |
4. High background signal in qPCR or NG-seq analysis. | Genomic DNA has become highly fragmented due to harsh treatment of samples. | Always use the Rabbit (DA1E) mAb IgG XP® Isotype Control (CUT&RUN) #66362 negative control antibody to determine background signal in the CUT&RUN assay. |
To minimize DNA fragmentation, avoid vigorous vortexing and introduction of bubbles during resuspension of cells. | ||
Genomic DNA has become highly fragmented due to cell stress and lysis. | Be sure to prepare cells at room temperature and as quickly as possible to minimize cell stress. Wash all cells in one vial to minimize cell loss (Section I, Steps 7-16). | |
Digestion is not performed at 0°C and chromatin is over-digested. | Digestion should be performed in an ice-water bath. Digestion at higher temperatures can significantly increase background signal. | |
Make sure to pre-cool cell samples and calcium chloride in an ice-water bath for 5 min prior to adding the calcium chloride and initiating the digest. Quickly mix the samples and place back in the ice-water bath. | ||
Large non-specific genomic DNA can also diffuse into the supernatant and contaminate the smaller fragments released by targeted digestion. | Do not incubate samples at 37°C for > 10 min and do not shake samples during incubation (Section III, Step 11). Ten minutes is sufficient for diffusion of digested fragments into the supernatant. | |
Large genomic DNA fragments can be removed by size-selection using AMPure® XP Beads or SPRIselect® Reagent Kit prior to qPCR analysis. | ||
For NG-seq analysis, shorten the PCR amplification time (10-15 sec) during library construction to exclude amplification of large DNA fragments. | ||
Too much antibody is used in the assay, resulting in non-specific binding and digestion. | If possible, be sure to use a CUT&RUN validated antibody at the recommended dilution. If not, ChIP-validated and IF-validated antibodies often work at their ChIP- and IF-recommended dilutions. You may need to titrate your antibody in the assay. |
posted October 2019
revised January 2021
Protocol Id: 1884
The CUT&RUN Assay Kit is 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.
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.
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.
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.
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