The interaction between protein and DNA constitutes the molecular basis of life activities and precisely regulates key biological processes, including gene transcription, DNA replication, and damage repair. The dynamic interaction between them runs through. In order to explore this regulatory mechanism, researchers developed ChIP-seq and CUT&Tag technologies, which are important tools for analyzing protein-DNA interactions. This article compares CUT&Tag and ChIP-seq technologies in epigenetics studies, covering their principles, signal-to-noise ratio, cell input requirements, workflows, costs, and offers key tips for choosing the right one based on research needs.

CUT&Tag vs ChIP-seq: Brief Introduction & Comparison

ChIP-seq

• Core Definition: A gold-standard epigenetic technique for mapping genome-wide binding sites of proteins to DNA, integrating immunoprecipitation with high-throughput sequencing.
• Key Principle: Relies on cross-linking proteins to DNA in situ, fragmenting chromatin, enriching target protein-DNA complexes with specific antibodies, and sequencing the associated DNA to identify binding regions.
• Typical Workflow: Includes cell cross-linking → chromatin fragmentation → immunoprecipitation (IP) → DNA purification → library construction → high-throughput sequencing → data alignment and peak calling.
• Primary Applications: Investigates histone modifications, transcription factor binding, and chromatin remodeling. Critical for studying gene regulation, cell differentiation, and disease-associated epigenetic alterations.
• Notable Characteristics: Offers broad target compatibility but requires high cell input and deeper sequencing to mitigate background noise from non-specific binding.

CUT&Tag

• Core Definition: A state-of-the-art epigenetic technique for profiling protein-DNA interactions in situ, combining antibody-mediated targeting with Tn5 transposase-driven cleavage and tagmentation.
• Key Principle: Uses target-specific antibodies to recruit protein A/G-Tn5 fusion transposase to binding sites; activated Tn5 cleaves adjacent DNA and appends sequencing adapters directly, eliminating the need for cross-linking or sonication.
• Typical Workflow: Involves sample permeabilization → primary/secondary antibody binding → pA/G-Tn5 recruitment → Mg²⁺-induced tagmentation → DNA purification → library amplification → high-throughput sequencing.
• Primary Applications: Enables high-resolution analysis of rare cell populations, individual cell epigenetics, and sensitive epitopes. Ideal for studies with limited starting material.
• Notable Characteristics: Delivers high signal-to-noise ratios (minimal background), requires low cell input, and has a fast workflow. Limitations include reliance on validated antibodies for target proteins.

Comparison between CUT&Tag and ChIP-seq

This article compares CUT&Tag and ChIP-seq technologies in epigenetics studies, covering their principles, signal-to-noise ratio, cell input requirements, workflows, costs, and offers key tips for choosing the right one based on research needs. More specific differences can be seen in the following contents.

Fundamental Principles: A Tale of Two Methodologies

ChIP-seq and CUT&Tag are the mainstream methods to explore the interaction between protein and DNA. There are essential differences between the two principles, which respectively represent the traditional in vitro detection and the new in situ detection technology path. Clarifying the principal differences is the basis for accurately selecting the technology and ensuring the reliability of the research.

Summary Details in the ChIP-seq Principle

ChIP-seq is a classic method to study the interaction between protein and DNA, which plays an important role in the field of epigenetics. Its principle is based on the natural binding state of protein and DNA in cells, and the precise location and analysis of the target protein binding DNA region can be achieved through a series of operations. The specific process is as follows:

• Cell culture and treatment: The target cell line is cultured to the logarithmic growth stage, and the cells are treated according to the experimental purpose, such as adding stimulating factors, drug treatment, etc., to induce the interaction between protein and DNA and simulate the molecular regulation process under specific physiological or pathological conditions.
• Crosslinking: Adding a crosslinking agent, such as formaldehyde, with a final concentration of about 1% to the cells, and gently rotating and incubating at room temperature for 10-15 minutes to crosslink and fix protein and DNA; Subsequently, glycine with a final concentration of 0.125M was added and incubated for 5 minutes to terminate the crosslinking reaction.
• Cell lysis and chromatin fragmentation: Wash cells with ice-cold PBS, add cell lysis solution, and incubate on ice for 10-15 minutes to rupture cells and release nuclei; Chromatin is broken into 200-500bp fragments by ultrasonic crushing (power 200-300W, 30 seconds each time, 30 seconds apart, 5-10 times in total) or enzyme digestion.
• Chromatin immunoprecipitation (IP): Use a specific antibody against the target protein (1-10 μg antibody is added to every 25 μg DNA), set an IgG antibody control and a control with magnetic beads only, and take 50 μL of chromatin as an input sample for later use. The first antibody was added to the sample and incubated at 4℃ for 1 hour. Add 60μL protein A/G magnetic beads, rotate at 4℃ overnight to bind the antibody-protein-DNA complex to the magnetic beads, collect the magnetic beads by centrifugation, remove the supernatant, and enrich the DNA fragments bound to the target protein.
• Elution and cross-linking: 120μL elution buffer was added to the protein A/G magnetic beads, and the DNA was eluted by slow vortex at 30℃ for 15 minutes. Transfer the supernatant after centrifugation, add 5M NaCl and RNase A, shake and incubate overnight at 65℃ to remove RNA impurities; Add protease K, shake and incubate at 60℃ for 1 hour to separate protein from DNA, and finally purify DNA by PCR purification kit or phenol-chloroform extraction.
• Establishing a library and sequencing: Constructing a sequencing library from the purified DNA fragments, and using a high-throughput sequencer. Compared with the reference genome, the position of the DNA fragment on the genome was determined, the binding site of the target protein was inferred, and the gene regulation mechanism was revealed. The minimum sequencing depth is 5-10M for transcription factor research and 20-40M for histone-modified broad-spectrum research.

Schematic diagram of ChIP-seq, CUT&RUN, CUT&Tag, and DAP-seq (Wang et al., 2023)

Detailed Explanation of the CUT&Tag Principle

CUT&Tag is a new technology to study the interaction between protein and DNA, which was innovatively developed by Steven Henikoff's team in 2019. It has the following characteristics and operation steps:

• A. Technical principles and advantages
  • a) Using antibody-mediated targeted recognition and the DNA shearing-labeling function of Tn5 transposase, the high-resolution detection of protein binding sites or epigenetic modification was realized, which brought a breakthrough for epigenetic research.
  • b) Taking living cells, nuclei, or fixed tissues as starting materials, sample fixation is usually optional, and chromatin can maintain its original structure and functional state under natural conditions, avoiding the destruction of chromatin structure by chemical crosslinking in the traditional technology, and more truly reflecting the interaction between protein and DNA.
• B. Key initial step: sample marking
  • a) A specific primary antibody (for target protein or histone modification, such as H3K27me3) is combined with the target region in the permeabilized nucleus, and the primary antibody can specifically recognize and bind the target protein.
  • b) A secondary antibody with an Fc fragment binding domain is added to connect protein A or protein G to form a cascade amplification system to enhance the specificity and intensity of the signal.
• C. Core link-positioning and cutting
  • a) Protein A/G (PA-Tn5) fused with TN5 transposase is added, and the transposase binds to the antibody complex in the target region.
  • b) After adding Mg²⁺ into the system to activate Tn5 transposase, it performs double-stranded cleavage on DNA near the antibody binding site, and at the same time adds sequencing linkers at both ends of the cleavage site to complete the tagging process. This design avoids the complicated steps of chromatin fragmentation and immunoprecipitation in the traditional technology, simplifies the experimental process, reduces the background noise, and improves the specificity and sensitivity of the experiment.
  • c) Library construction: The cut DNA fragments have library connectors directly, which can be used in high-throughput sequencing by simple amplification. This one-step library construction method saves time and cost, reduces the errors that may be introduced in the experimental operation, and improves the repeatability of the experiment.

The histogram diagram showed the annotation of peaks for the H3K4me3 histone modification from CUT&Tag and ChIP data (Tao et al., 2020)

The Critical Trade-off: Signal-to-Noise vs. Universality

In epigenetics research, technology selection often needs to be balanced between core performance, and the game between signal-to-noise ratio and universality is the core focus of the comparison between ChIP-seq and CUT&Tag technologies. The former, as a classic method, covers more research needs with its extensive antibody adaptability and mature application scenarios, but is limited by background interference. The latter achieves high SNR data output with low background advantage, but its application scope is restricted by the antibody and sample type. This key trade-off between the two directly determines the quality of experimental data and the feasibility of research, and is the primary consideration for technical selection of scientific researchers.

Signal Advantage of CUT&Tag

CUT&Tag technology has outstanding performance in signal acquisition, and its unique technical principle makes it have obvious advantages of low background noise and high signal-to-noise ratio. Although the traditional ChIP-seq technology realizes the separation of protein-DNA complex by formaldehyde crosslinking and ultrasonic crushing, it also brings a series of problems:

• Formaldehyde cross-linking: When formaldehyde cross-links the target protein with DNA, it may also trigger nonspecific protein-DNA cross-linking, thus increasing the background noise.
• Ultrasonic fragmentation: Ultrasonic fragmentation may destroy the natural structure of chromatin, resulting in the false release of DNA fragments that were originally weakly bound to protein, which interferes with the detection of real signals.

In contrast, CUT&Tag technology cleverly circumvents these problems:

• Operating conditions: CUT&Tag technology operates under natural conditions without formaldehyde crosslinking, thus avoiding the background interference caused by nonspecific crosslinking. In the study of histone modification of mouse embryonic stem cells, researchers used CUT&Tag and ChIP-seq techniques to detect it. The results showed that the signal peak detected by CUT&Tag was sharper and the background noise was lower, which could clearly distinguish the enrichment of H3K4me3 in the gene promoter region. ChIP-seq can detect the H3K4me3 signal, but the background noise is high and the signal peak is wide, so it is difficult to locate the binding site of H3K4me3 accurately.
• individual cell level research: In individual cell level research, due to the scarcity of cells and weak signal intensity, the technical sensitivity is extremely high. CUT&Tag technology can effectively detect transcription factors and histone modification signals in individual cells, which provides a powerful tool for the study of individual cell epigenetics. However, ChIP-seq technology has a poor detection effect at the individual cell level because of high background noise, and it is difficult to obtain reliable results.

Universality and Challenge of ChIP-seq

ChIP-seq technology, as a classical method to study protein-DNA interaction, is highly universal in the extensive study of protein-DNA interactions. After years of development and improvement, this technology has established a set of mature experimental procedures and data analysis methods, which are suitable for the study of protein-DNA interactions of various species and types. ChIP-seq technology can provide reliable experimental results, whether it is the study of common model organisms (such as mice and fruit flies) or the exploration of non-model organisms. ChIP-seq technology is also widely used in the study of the interaction between transcription factors, histone modification, chromatin remodeling complexes, and DNA.

However, ChIP-seq technology faces severe challenges in dealing with non-specific binding and high background problems, which are embodied in the following aspects:

• Immunoprecipitation step: The binding of the antibody to the target protein is not completely specific, and it may cross-react with some non-target proteins, thus leading to the enrichment of non-specific DNA fragments.
• Ultrasonic fragmentation process: Because it is difficult to accurately control the degree and position of fragmentation, some DNA fragments unrelated to the target protein may be produced, further increasing the background noise.

The above nonspecific binding and high background problems will not only interfere with the detection of real signals but also lead to wrong experimental conclusions. When studying some transcription factors with low abundance, due to their low expression in cells, the signal is easily drowned out by background noise, which makes it difficult to accurately detect their binding sites by ChIP-seq technology. In order to overcome these problems, researchers usually need to increase the sequencing depth to improve the sensitivity of signal detection, but this will increase the experimental cost and the difficulty of data analysis.

Reproducibility of H3K4me3 and H3K27me3 ChIP-seq with the new method (Gilfillan et al., 2012)

Input Requirements and Practical Considerations

Sample input is the core premise to determine the applicability of technology, which is directly related to experimental feasibility, data reliability, and research cost. The significant difference between CUT&Tag and ChIP-seq in cell input requirements makes them form complementary application scenarios for different sample types (such as rare cells, limited clinical samples, and conventional cell lines).

Low Cell Input Advantage of CUT&Tag

CUT&Tag technology has shown remarkable advantages in the requirement of cell input, and its demand for initial cell quantity is extremely low, only 100-100,000 cells are needed, and effective detection can be realized even at the individual cell level. This characteristic makes it have unparalleled advantages in studying rare cell populations or clinical samples.

• In the field of neuroscience, it is extremely difficult to obtain neuron cells, and they are rare. The traditional ChIP-seq technology is difficult to play a role in the study of neuronal cells because of its high demand for cell quantity, while the CUT&Tag technology provides the possibility for the study of neuronal cells.
• In tumor research, circulating tumor cells (CTCs), as an important marker of tumor metastasis and recurrence, have important research value. However, the content of CTCs in blood is extremely low, with only a few to dozens of CTCs per milliliter of blood. With the advantage of low cell input, CUT&Tag technology can analyze a very small amount of CTCs.

ChIP-seq Input Requirements and Limitations

ChIP-seq technology usually needs 10 to millions of cells as starting materials. This is because the experimental flow of ChIP-seq involves many steps that are prone to cause sample loss, such as ultrasonic disruption, IP, and so on. In order to obtain enough DNA fragments bound to the target protein, a large number of starting cells need to be used. ChIP-seq can play an effective role in obtaining a large number of cell samples, such as common cell lines.

However, when it is difficult to obtain samples, the demand for ChIP-seq for a large number of cells becomes a significant limitation. An insufficient sample size will also have a serious impact on the experimental results. If the number of initial cells is too small, enough DNA fragments bound to the target protein may not be obtained, resulting in insufficient signal intensity, and it is difficult to accurately detect protein-DNA binding sites. At the same time, a low sample size will increase experimental error and uncertainty, and reduce the reliability of experimental results.

Schematic of scCuT&Tag applied to cultured cells and tissues in different species (Fu et al., 2023)

Making the Choice: Which Technique is Right for Your Project

The choice of CUT&Tag and ChIP-seq is by no means arbitrary, but a key decision that needs to be closely related to the core requirements of the project. The former highlights its advantages with low cell input and high signal-to-noise ratio, while the latter is based on mature universality, and both have their own limitations. If technology and research objectives are misaligned, it will easily lead to waste of resources, data distortion, and even experimental failure. Therefore, it is very important to establish a demand-oriented selection framework to improve research efficiency and ensure the reliability of results.

Select According to Sample Characteristics

In the study of protein-DNA interaction, the characteristics of samples are an important basis for technology selection, and the number, type, and difficulty of obtaining samples will significantly affect the experimental results.

• Cell number: For samples with limited cell numbers, such as circulating tumor cells (CTCs) and hematopoietic stem cells, CUT&Tag technology has obvious advantages. The content of CTCs in blood is extremely low, and the traditional ChIP-seq technology requires a high amount of cells, so it is difficult to analyze a small amount of CTCs, while the CUT&Tag technology only needs 100-100,000 cells, even at the individual cell level, which is helpful to reveal the mechanism of tumor metastasis and provide a new target for tumor diagnosis and treatment.
• Sample type: Sample type also affects technology selection. For samples that are sensitive to cross-linking, such as some fragile protein-DNA complexes, CUT&Tag technology can operate under natural conditions without cross-linking, which can better preserve the natural state of samples. When studying transcription factors sensitive to cross-linking, the cross-linking step of ChIP-seq may destroy the binding and lead to inaccurate results, but the CUT&Tag technology can avoid this problem.

Choose based on the Research Objectives

Research goal is one of the core factors that determine the choice of technology. Different research objectives have different technical requirements. Only by choosing the technology that matches the research objectives can accurate and reliable experimental results be obtained.

• If a high-resolution protein-DNA interaction map is needed and background noise is strictly required, CUT&Tag technology is the first choice. The advantages of a high signal-to-noise ratio and low background noise can meet the demand.
• ChIP-seq technology can be used as a reliable method if high-quality chip-level antibodies are available for the target protein, and there is no special concern about the cross-linking and fixing steps, such as studying common transcription factors or histone modification.
• ChIP-seq technology is still an indispensable tool for research that needs extensive research on protein-DNA interaction and requires high technical versatility.
• Other influencing factors

Other Considered Factors

In addition to the sample characteristics and research objectives, there are other factors that will affect the technology selection:

• Availability of antibody: Both ChIP-seq and CUT&Tag technologies rely on specific antibodies to recognize the target protein. ChIP-seq technology can be used as a reliable method if there are high-quality chip-level antibodies and there is no special concern about the cross-linking and immobilization steps. However, if antibody resources are limited or if you want to avoid the potential impact of cross-linking, CUT&Tag technology is a more flexible alternative.
• Cost and experimental period: CUT&Tag technology has a short experimental period and relatively low cost, which is suitable for the scene of rapid data acquisition, especially for research with limited samples. ChIP-seq technology has a long experimental period and relatively high cost, which makes it more suitable for mature experimental platforms with sufficient samples.

The development of cleavage under targets and tagmentation technologies (Xiong et al., 2024)

Conclusion

To sum up, CUT&Tag and ChIP-seq, as the core technologies for analyzing protein-DNA interaction in epigenetics research, should be comprehensively judged around key dimensions such as sample characteristics, data requirements, experimental cost, etc.

• Under the demand of low cell volume and high signal-to-noise ratio, CUT&Tag has obvious advantages in a one-day process and low background.
• However, in the scenario of limited antibody compatibility and relying on mature optimization schemes, ChIP-seq still has irreplaceable application value.

They are not substitutes, but complementary tools for different research goals. Whether focusing on the fine analysis of rare clinical samples or carrying out large-scale verification research of known targets, accurately matching technical characteristics with scientific problems is the key to promoting the in-depth analysis of the epigenetic mechanism.

In the future, with the continuous iteration of technology, the process optimization and application boundary expansion of the two methods will further provide more flexible and efficient technical support for epigenetics research and help researchers explore more unknowns in the fields of gene regulation and disease mechanisms.

References

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3. Gilfillan GD, Hughes T, Sheng Y, et al. "Limitations and possibilities of low cell number ChIP-seq." BMC Genomics. 2012 13 :645.
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