CUT&Tag Overview

Like CUT&RUN, Cleavage Under Targets & Tagmentation (CUT&Tag) is a faster, more cost-friendly alternative to ChIP-seq. Compared to ChIP-Seq, CUT&Tag profiles chromatin with less sample, a simplified workflow, lower sequencing costs, and improved signal-to-noise due to lower backgrounds. CUT&Tag is faster than CUT&RUN because most of the library prep occurs in vivo, delivers equivalent data to CUT&RUN, and enables single-cell CUT&Tag.

Cell Signaling Technology^® CUT&Tag reagents and kits are validated with the same rigor that is applied to other products to ensure performance. CST also uses these same reagents to validate CUT&Tag antibodies, ensuring performance and reliability every time.

What Makes CUT&Tag Different

CUT&Tag and CUT&RUN can each be used to profile native chromatin, whereas ChIP and ChIP-seq traditionally utilize cross-linked chromatin. For both CUT&Tag and CUT&RUN, the cellular membrane is permeabilized so the primary antibody can enter the nucleus through the nuclear pore, where they bind to the target of interest. The appropriate pAG-enzyme binds to the antibody and, when activated, cuts the DNA on either side of where the antibody is bound.

CUT&RUN and CUT&Tag are also both compatible with Drosophila spike-in normalization controls. The Drosophila spike-in nuclei are added at the beginning of the experiment; the corresponding H2Av antibody is then added later during the primary antibody incubation step.

Experimental Workflow with Drosophila Spike-in Control

The CUT&Tag workflow (left) delivers robust protein-DNA interaction data while decreasing DNA library prep time.

How does CUT&Tag differ from CUT&RUN?

• CUT&Tag utilizes a secondary incubation step after the primary antibody incubation step to boost signal strength.
• CUT&Tag is performed under high salt conditions. Both methods are compatible with histone analysis. However, CUT&Tag is less compatible with transcription factors and cofactors because high salt conditions can interfere with target protein-DNA interactions. This is particularly true for less abundant or weakly bound targets.
• CUT&RUN uses Ca2⁺-activated pAG-MNase to cleave the DNA while CUT&Tag uses Mg2+-activated pAG-Tn5 to cleave the DNA. The Tn5 is charged with Illumina adaptors that are added to the chromatin during the cleavage process.
• CUT&Tag permeabilizes both the cellular and nuclear membrane after tagmentation to completely solubilize the CUT&Tag DNA. This leads to the presence of both tagmented DNA and genomic DNA in the final DNA sample, making CUT&Tag DNA incompatible with qPCR. If qPCR is desired, we recommend performing CUT&RUN.
• CUT&Tag skips the in vitro adaptor ligation step required for CUT&RUN, allowing you to skip directly to PCR amplification of your DNA library for sequencing–saving you precious time.
• CUT&Tag's time savings is cumulative, meaning you'll see even more time savings as you scale up the number of samples you have.

CUT&Tag delivers:

Whole-Workflow Spike-In Normalization Control is Now Available for CUT&Tag

CST offers two CUT&Tag Drosophila Spike-In Controls Kits that are compatible with CUT&Tag Assay Kit #77552:

Drosophila Spike-in Normalization Increases Confidence in Drug Response Data

In the normalized data, the tazemetostat‑treated sample shows reduced H3K27me3 and Ezh2 signals, consistent with EZH2 inhibition, while H3K4me3 and H3K27ac signals remain comparable. This demonstrates how the Drosophila spike-in normalization strategy increases confidence that observed changes in global histone marks represent true biological responses, rather than technical variation.

Drosophila Spike-In Control Kit for CUT&Tag (Rabbit)

CUT&Tag was performed with 100,000 MCF7 cells, with or without treatment of 1 μM Tazemetostat for 6 d (as indicated), and either Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit Monoclonal Antibody #9733, Ezh2 (D2C9) Rabbit Monoclonal Antibody #5246, Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751, or Acetyl-Histone H3 (Lys27) (D5E4) Rabbit Monoclonal Antibody #8173, using CUT&Tag Assay Kit #77552 and the Drosophila Spike-In Control Kit for CUT&Tag (Rabbit). DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across the HOXA gene cluster. After normalization, both H3K27me3 and EZH2 signal is significantly reduced following EZH2 inhibitor treatment, consistent with decreased H3K27me3 levels. In contrast, both H3K4me3 and H3K27Ac signals remain comparable, or are slightly increased, as this drug does not directly affect these marks. Instead, reduced H3K27me3 occupancy may permit increased H3K4me3 or H3K27Ac binding at the same loci.

In the normalized data, the tazemetostat‑treated sample shows a reduced H3K27me3 signal, consistent with EZH2 inhibition, while H3K4me3 signal remains comparable. This demonstrates how the Drosophila spike-in normalization strategy increases confidence that observed changes in global histone marks represent true biological responses, rather than technical variation.

Drosophila Spike-In Control Kit for CUT&Tag (Mouse)

CUT&Tag was performed with 100,000 MCF7 cells, with or without treatment of 1 μM Tazemetostat for 6 d (as indicated), and either mouse H3K27me3 antibody or mouse H3K4me3 antibody, using CUT&Tag Assay Kit #77552 and the Drosophila Spike-In Control Kit for CUT&Tag (Mouse). DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across the HOXD gene cluster and ACTB gene, known targets of H3K27me3 and H3K4me3, respectively. After normalization, H3K27me3 signal is significantly reduced following EZH2 inhibitor treatment, consistent with decreased H3K27me3 levels. In contrast, H3K4me3 signal remains comparable, as this drug does not affect H3K4me3.

Accurate Signal Normalization Across Cell Titrations with Drosophila Spike-In Control

Drosophila spike‑in normalization ensures that CUT&Tag signal intensities scale appropriately with starting cell number. In the cell titration experiment below, the normalized data shows a strong positive correlation with signal strength across the MYC gene and starting cell number, demonstrating successful correction of sample input variability.

Drosophila Spike-In Control Kit for CUT&Tag (Rabbit)

CUT&Tag was performed with 200,000, 100,000, 50,000, or 25,000 HeLa cells (as indicated) and CTCF (D1A7) Rabbit Monoclonal Antibody #3417, using CUT&Tag Assay Kit #77552 and the Drosophila Spike-In Control Kit for CUT&Tag (Rabbit). DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across MYC, a known target gene of CTCF. After normalization, signal strength shows a positive correlation with the starting cell number.

The normalized data shows a strong positive correlation with signal strength across the GAPDH gene and starting cell number, demonstrating successful correction of sample input variability.

Drosophila Spike-In Control Kit for CUT&Tag (Mouse)

CUT&Tag was performed with 100,000, 50,000, or 25,000 HeLa cells (as indicated) and a mouse H3K4me3 antibody, using CUT&Tag Assay Kit #77552 and the Drosophila Spike-In Control Kit for CUT&Tag (Mouse). DNA libraries were prepared using CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows binding across GAPDH, a known target gene of H3K4me3. After normalization, signal strength shows a positive correlation with the starting cell number.

When to Use CUT&Tag vs CUT&RUN

Both CUT&Tag and CUT&RUN help you unravel protein-DNA interactions when you are short on time and/or sample.

Use the table below to figure out which method is the right one for you.

CUT&Tag is compatible with next-generation sequencing (NGS) and is ideal for studying how histone modifications regulate chromatin binding when sample and time are limited. It can also be used to study transcription factors when the antibody used is validated specifically for CUT&Tag by you or a commercial vendor like CST.

Order the CST^® CUT&Tag Assay Kit #77552 and the relevant Drosophila Spike-In Control Kit to get all the reagents you need for your CUT&Tag experiment or purchase just the reagents you need a la carte. All CUT&Tag reagents are stringently validated in-house to ensure you’ll get high-quality reagents every time. You can also choose from an ever-expanding list of CST CUT&Tag-validated antibodies.

Comparable Data, Faster Results

CUT&Tag reagents give you the same high-quality data possible with CUT&RUN in half the library prep time.

Analyzing Histone Modifications with CUT&Tag

Generate equivalent data to ChIP-seq and CUT&RUN with the CUT&Tag Assay Kit #77552, CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415, or a la carte products when studying histone modifications like Tri-Methyl-Histone H3 (Lys4) or Tri-Methyl-Histone H3 (Lys27). Go from cells to library DNA in 1-2 days with 100,000 starting cells.

H3K4me3

ChIP-seq, CUT&RUN, or CUT&Tag assays were performed with HCT 116 cells and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552, respectively. DNA libraries were prepared using the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples, and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figure shows binding across chromosome 12 (upper), including GAPDH (lower), a known target gene of H3K4me3.

H3K27me3

ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with NCCIT cells and Tri-Methyl-Histone H3 (Lys27) (C36B11) Rabbit Monoclonal Antibody #9733, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K27me3 across chromosome 10 (upper), including the PAX2 gene (lower).

Analyzing Transcription Factors and Cofactors with CUT&Tag

Generate equivalent data to ChIP-seq and CUT&RUN with the CUT&Tag Assay Kit #77552, CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415, and CUT&Tag-validated antibodies when studying transcription factors or cofactors like Nanog, Estrogen Receptor α, and JARID2. Go from cells to library DNA in 1-2 days with 100,000 starting cells.

Nanog

ChIP-seq, CUT&RUN, or CUT&Tag assays were performed with F9 cells and Nanog (D2A3) Rabbit Monoclonal Antibody (Mouse Specific) #8822, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552, respectively. DNA libraries were prepared using the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples, and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figure shows binding across chromosome X (upper), including Xist (lower), a known target gene of Nanog.

Estrogen Receptor α

ChIP-seq, CUT&RUN, or CUT&Tag assays were performed with MCF7 cells grown in phenol red-free medium and 5% charcoal-stripped FBS for 4 days then treated with β-estradiol (10 nM) for 45 minutes and Estrogen Receptor α (D8H8) Rabbit Monoclonal Antibody #8644, using the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, and CUT&Tag Assay Kit #77552, respectively. DNA libraries were prepared using the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figure shows binding across chromosome 21 (upper), including TFF1 (lower), a known target gene of Estrogen Receptor α.

JARID2

CUT&Tag, CUT&RUN and ChIP-seq assays were performed with NCCIT cells and JARID2 (D6M9X) Rabbit Monoclonal Antibody #13594, using the CUT&Tag Assay Kit #77552, the CUT&RUN Assay Kit #86652, or the SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005. DNA libraries were prepared using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples and the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples. The upper panel compares enrichment around HOXA genes, while the lower panel compares enrichment around HOXD genes, both are known target genes of JARID2.

Analyzing Tissue Samples with CUT&Tag

You can also analyze histone marks in tissue samples using CUT&Tag. We recommend using the CUT&RUN Assay Kit #86652 if you are analyzing transcription factors or cofactors in tissues.

Brain Tissue

ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with 25 mg (for ChIP-seq) or 1 mg (for CUT&RUN and CUT&Tag) of fresh mouse brain tissue and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, or CUT&Tag Assay Kit #77552. DNA libraries were prepared using the DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K4me3 across chromosome 6 (upper), including Gapdh (lower), a known target gene of H3K4me3.

Liver Tissue

ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with 25 mg (for ChIP-seq) or 1 mg (for CUT&RUN and CUT&Tag) of fresh mouse liver tissue and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, or CUT&Tag Assay Kit #77552. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K4me3 across chromosome 6 (upper), including Gapdh (lower), a known target gene of H3K4me3.

Heart Tissue

ChIP-seq, CUT&RUN, and CUT&Tag assays were performed with 25 mg (for ChIP-seq) or 1 mg (for CUT&RUN and CUT&Tag) of fresh mouse heart tissue and Tri-Methyl-Histone H3 (Lys4) (C42D8) Rabbit Monoclonal Antibody #9751, using SimpleChIP^® Plus Enzymatic Chromatin IP Kit (Magnetic Beads) #9005, CUT&RUN Assay Kit #86652, or CUT&Tag Assay Kit #77552. DNA libraries were prepared using DNA Library Prep Kit for Illumina Systems (ChIP-seq, CUT&RUN) #56795 for ChIP-seq and CUT&RUN samples and CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415 for CUT&Tag samples. The figures show enrichment of H3K4me3 across chromosome 6 (upper), including Gapdh (lower), a known target gene of H3K4me3.

Sequencing Success Even When Bioanalyzer or TapeStation System Signal Is Low

Purified CUT&Tag DNA can be quantitated using platforms like the Thermo Fisher Scientific NanoDrop or Qubit Fluorometric Quantification system before being QC’d with platforms like the Agilent Bioanalyzer or TapeStation system prior to NGS. It should be noted that the calculated DNA library yield may differ depending on the quantitation method used.

You can still successfully sequence your DNA library even if you see very weak or no visible peaks in the Agilent Bioanalyzer or TapeStation system profile regardless of the CUT&Tag reagents used because CUT&Tag baselines are lower than ChIP-seq and CUT&RUN. Therefore, we recommend proceeding with sequencing because it is still possible to obtain sequencing data with high genomics signal even if the Bioanalyzer or TapeStation system signal is low.

Key tips for low-yield libraries:

• Use the Right Tools: For the most accurate assessment, we recommend using the High Sensitivity ScreenTape Assay rather than the Standard version. Its optimized chemistry is better at picking up the subtle signals of a CUT&Tag library.
• Histone Modifications vs Transcription Factors: While histone modifications usually show a clear signal, libraries for transcription factors or cofactors often show very weak or even no visible peaks.
• Trust the Process: Even with a low signal, these libraries frequently produce high-quality NGS results with strong mapping rates and clear binding peaks.
• Pooling for Success: If the Bioanalyzer or TapeStation cannot determine your average library size, we recommend using an estimated size of 900 bp. This allows you to intentionally pool low-yield libraries at a slightly higher proportion to ensure great sequencing results.

For a deeper dive into managing low yields, read the blog: CUT&Tag DNA Library Yield: What to Do if it's Too Low to Detect.

Bioanalyzer System Profiles for Three Different CUT&Tag Libraries

CUT&Tag was performed with HCT 116 cells and TCF4/TCF7L2 (C48H11) Rabbit Monoclonal Antibody #2569, with several sources of loaded pAG-Tn5. The amount of each enzyme used was based on the manufacturer’s recommendation. DNA libraries were prepared using the CUT&Tag Dual Index Primers and PCR Master Mix for Illumina Systems #47415. The figure shows the Bioanalyzer system profile of the CUT&Tag library DNA. The same DNA libraries were sequenced and their NGS tracks are shown below.

NGS Tracks of Three Different CUT&Tag Libraries

The DNA libraries analyzed with the Bioanalyzer system were sequenced and NGS tracks are shown. NGS data shows equivalent binding across chromosome 8 (upper), including MYC (lower), a known target gene of TCF4.

Alternatively, you can QC your CUT&Tag DNA library with qPCR by using primers against known positive and negative gene loci on the library DNA to determine the enrichment of chromatin fragments before NGS.

CUT&Tag Product Options

CST CUT&Tag kits and a la carte reagents are validated in-house by our Epigenetics Application Team, comprised of bench scientists and subject-matter experts, to ensure the reagents work when they arrive in your lab. Find the right solution for your workflow below.

References

1. Kaya-Okur HS, Wu SJ, Codomo CA, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019;10(1):1930. Published 2019 Apr 29. doi:10.1038/s41467-019-09982-5
2. Egan, B. et al. An Alternative Approach to ChIP-Seq Normalization Enables Detection of Genome-Wide Changes in Histone H3 Lysine 27 Trimethylation upon EZH2 Inhibition. (2016) PLoS ONE 11, e0166438
3. Taruttis, F. et al. External calibration with Drosophila whole-cell spike-ins delivers absolute mRNA fold changes from human RNA-Seq and qPCR data. (2017) Biotechniques 62, 53-61 Pubmed 28193148

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