In the field of biomedical research, the study of chromatin accessibility and protein-DNA interaction is very important for understanding the regulation mechanism of gene expression. As a new epigenetic research method, CUT&Tag technology has quickly become an important tool to analyze chromatin state and transcription factor binding sites with its advantages of low cell start, high resolution, and low background. However, in the actual operation process, the complexity of the CUT&Tag experiment makes the experimental results easily disturbed by many factors, and technical bottlenecks may occur in all links from sample preparation to data processing.

Therefore, mastering systematically the optimization strategy and troubleshooting method of the CUT&Tag experiment will not only help to improve the success rate of the experiment, but also provide a solid guarantee for researchers to obtain high-quality and reliable data. This paper will focus on the whole process of the CUT&Tag experiment, deeply discuss the optimization scheme of key steps, and put forward detailed solutions to common problems, which will help scientific researchers overcome experimental problems and promote the in-depth development of epigenetics research.

Critical Pre-Experimental Considerations

Before the CUT&Tag experiment is carried out, a key pre-consideration is needed. The effectiveness of the antibody, the rationality of the cell preparation, and the scientificity of experimental control are the core prerequisites to determine whether the experiment can successfully obtain accurate and reliable epigenetic data, which directly affects the interpretation and application of subsequent results.

Antibody Verification: Core Premise of Experiment

Antibody, as the core molecule of the target antigen in the CUT&Tag experiment, has its specificity and applicability, which are the key parameters to determine the reliability of experimental results. Compared with the ChIP experiment, the CUT&Tag technology adopts the in-situ detection strategy, which requires that the antibody still has high antigen recognition ability in the natural chromatin conformation. This technical feature puts forward special requirements for the selection of antibodies, that is, the antibodies verified by CUT&Tag or CUT&RUN experiments should be preferred, while the antibodies verified by ChIP experiments are not necessarily suitable for the CUT&Tag system.

From the point of view of the action mechanism, some antibodies verified by ChIP rely on the stable binding environment formed by formaldehyde crosslinking when recognizing antigens. In the non-crosslinked system of the CUT&Tag experiment, the binding affinity between such antibodies and antigens decreased significantly, which led to a decrease in enrichment efficiency. In addition, the process of cell permeabilization may change the spatial conformation of chromatin, so that some epitopes recognized by antibodies are sterically hindered or chemically modified, and then the specific binding between antibody and antigen is affected.

Cell Preparation: Optimization of Adapting to Different Cells

Cell permeability is the key step of the CUT&Tag experiment. It is necessary to maintain the integrity of the nucleus and let antibodies and subsequent reagents enter the nucleus. Different cell types (such as primary cells and cell lines) have different characteristics of the cell membrane and nuclear membrane, so it is necessary to optimize the permeability conditions.

• Cell line: Usually, the buffer containing 0.1% Digitonin saponin is used, and it can be effectively penetrated by incubating on ice for 5-10 min, with little damage to cells.
• Primary cell: The primary cell membrane is more fragile, which can reduce the concentration of digitalis saponin to 0.05% and shorten the incubation time to 3-5 min. At the same time, the cell morphology should be closely observed to avoid nuclear rupture caused by excessive permeability.

Experimental Control: Measures to Ensure the Reliability

Setting perfect control is the key to eliminating experimental interference and ensuring the specificity of the results.

• Antibody-free control: Detecting nonspecific signals of the experimental system itself, such as spontaneous cleavage of Tn5 transposase.
• IgG control: Use unrelated antibodies of the same type as the primary antibody to evaluate the nonspecific binding of the antibodies.
• Positive control antibody: Such as H3K4me3 (widely found in the promoter region of active genes), which should be able to generate clear and repeatable signals in the CUT&Tag experiment to verify the effectiveness of the experimental system.

The principle of CUT&Tag and CUT&Tag variants (Fu et al., 2023)

The Wet-Lab Protocol: Optimizing Each Step

The wet experiment process of CUT&Tag is the core link of technical success, and the parameter optimization of each step is very important. From cell permeability to labeling reaction, the conditions of each step (such as reagent concentration, incubation time, etc.) need to be accurately adjusted to ensure the efficient acquisition of target DNA fragments and the quality of subsequent libraries.

Cell Permeability: Balancing Permeability and Cell Integrity

The concentration of digitalis saponin and incubation time are the core parameters of permeability. The optimal conditions can be determined by gradient experiment: setting different digitalis saponin concentrations (such as 0.02%, 0.05%, 0.1%, 0.2%) and incubation time (such as 2 min, 5 min, 10 min, 15 min), then observing the cell morphology (the cell nucleus is intact and the cell membrane is permeable) through a microscope, or the intensity and specificity of the target signal in subsequent experiments, and screening out the best combination.

Antibody Incubation: Specificity and Binding Efficiency

Too high antibody concentration can easily lead to nonspecific binding, and too low antibody concentration can lead to insufficient signal. It is necessary to optimize the antibody concentration gradient, usually starting from 1 μg/mL, setting gradients of 0.5 μg/mL, 1 μg/mL, 2 μg/mL and 5 μg/mL, and incubating overnight at 4℃ or at room temperature for 2-4 h, and determining the optimal concentration through the ratio of signal intensity of target peak to background noise in subsequent experiments. At the same time, the incubation time also needs to be optimized. Too long may increase nonspecific binding, and too short may lead to insufficient binding. Generally, incubation at room temperature for 2-4 h is better.

PA-Tn5 Treatment: Maintaining Transposase Activity

PA-Tn5 fusion protein is sensitive to temperature, pH, and other conditions, so it needs to be diluted and preserved strictly according to the instructions. When diluting, use a special dilution buffer to avoid repeated freezing and thawing. Usually, dilute it now and put it on ice after dilution. The operation time should not be too long to maintain its activity.

Tagging: Regulating the Size of DNA Fragments

Mg²⁺ concentration and reaction time are the keys to regulating the transposase cleavage activity and DNA fragment size of Tn5. Excessive Mg²⁺ concentration will lead to excessive cleavage and produce too short DNA fragments. If the concentration is too low, the cutting is insufficient and the fragment is too long. The concentration gradient of 1 mM, 2.5 mM, 5 mM, and 10 mM of Mg²⁺ can be set, and the incubation time gradient of 30 min, 60 min, and 90 min at 37℃ can be used to determine the optimal conditions through the fragment size distribution of the subsequent library (ideal range is 200-1000 bp).

Reproducibility and efficiency of CUT&Tag (Kaya-Okur et al., 2019)

From Library to Data: Post-Tagmentation Steps

After tagging, a series of subsequent treatments is needed to obtain a high-quality library with a measurable sequence. This stage covers key steps such as DNA fragment extraction, library amplification, and quality control, and the operation accuracy of each step directly affects the reliability and availability of the final sequencing data.

DNA Fragment Extraction: Recovery of Target Fragments

After the labeling reaction is completed, the DNA fragment with the sequencing linker should be released and purified. A PCR purification kit or the magnetic bead method can be used for purification. The magnetic bead method needs to optimize the ratio of magnetic beads to samples. Usually, the ratio of magnetic beads is 1.8×, which can effectively recover more than 200 bp fragments and remove impurities such as primer dimer.

Library amplification: Avoiding Over-amplification

PCR amplification is used to add complete sequencing connectors and barcodes. Too many cycles will lead to over-amplification, repeated sequences, and increased background noise. Too few cycles will lead to insufficient library output. It is necessary to optimize the number of PCR cycles, monitor the amplification process by real-time quantitative PCR (qPCR), or set the cycle number gradient of 10, 12, 14, and 16, detect the library concentration and fragment distribution after amplification, and select the number of cycles (usually 12-16 cycles) that can ensure the yield without over-amplification.

Library Quality Control: Ensuring Sequencing Quality

Using Bioanalyzer or TapeStation to control the quality of the library and detect the fragment size distribution and concentration of the library. The ideal CUT&Tag library fragment size should be mainly distributed in 200-1000 bp, and there is no obvious primer dimer peak. The concentration should meet the requirements of the sequencing platform. If the fragment size distribution is abnormal or the concentration is insufficient, the previous experimental steps should be optimized.

Examples of Successful and Failed Libraries on an Agilent Bioanalyzer 2100 and Caliper LabChip GX (Xu et al., 2020)

Troubleshooting Common CUT&Tag Pitfalls

Although the CUT&Tag technology brings convenience to epigenetics research, it is easy to encounter problems such as low library yield, high background noise, and no signal in the experiment. If these problems are not checked in time, the experimental results will be seriously affected. This paper focuses on common problems, analyzes the causes and solutions, and helps the experiment to be carried out smoothly.

Low Library Yield

• A. Potential cause
  • a) Insufficient number of cells: As the source of target DNA, if the initial number of cells is lower than the threshold, it will directly lead to a decrease in the total amount of target DNA and a lack of raw materials for subsequent library construction. When the number of cells is insufficient, even if the efficiency of subsequent steps is normal, the final library output is difficult to reach the ideal level.
  • b) Low permeability: The Permeation process is the key link for the antibody and pA-Tn5 to enter the nucleus. If the permeability efficiency is not good, the antibody can't fully combine with the target antigen in the nucleus, and pA-Tn5 can't effectively enter the nucleus to play the role of transposase, which will affect the initial reaction of library construction and lead to a decrease in yield.
  • c) Inactivation of Tn5 transposase: The activity of Tn5 transposase is the core factor of DNA cleavage. Improper preservation (such as improper temperature and humidity) or wrong dilution process may cause the loss or decrease of transposase activity, resulting in low cutting efficiency and affecting library construction.
• B. Solution strategy
  • a) Ensure the number of cells: strictly control the number of initial cells, at least 100 cells, and recommend using 1000-10000 cells to provide sufficient target DNA raw materials.
  • b) Optimize the permeability conditions: observe the morphological changes of cells after permeability by microscope, or carry out pre-experiments to test the effects under different permeability conditions, and choose the best permeability scheme to ensure that antibodies and pA-Tn5 can effectively enter the nucleus.
  • c) Ensure transposase activity: carefully check the storage conditions (such as low temperature, dark, etc.) and dilution ratio of pA-Tn5. If the activity is suspected to be insufficient, replace the pA-Tn5 reagent in time.

High Background Noise

• A. Potential cause
  • a) Non-specific binding of antibody: When the specificity of the antibody is insufficient, it will bind to non-target antigen and guide Tn5 transposase to cut in a non-target area, resulting in a large number of non-specific DNA fragments and increasing background noise.
  • b) Inadequate washing: The residual non-specific binding antibody or pA-Tn5 will continue to trigger a non-specific reaction, producing additional DNA fragments in the subsequent steps, interfering with the experimental results and increasing the background signal.
  • c) Overdigestion: Excessive Mg²⁺ concentration or long reaction time in the reaction system will lead to excessive transposase activity of Tn5, excessive cleavage in non-target areas, and a large amount of non-specific DNA, resulting in increased background noise.
• B. Solution strategy
  • a) Optimizing antibody conditions: Screening more specific antibodies through experiments, and optimizing antibody concentration and incubation conditions to reduce nonspecific binding.
  • b) Increase the washing times: Increase the washing times from 3 times to 5 times to ensure that the non-specifically bound reagents are fully removed and reduce background interference.
  • c) Control of digestion conditions: Accurately optimize the concentration of Mg²⁺ and reaction time, and avoid non-specific products caused by excessive cutting while ensuring effective cutting of the target area.

Overview of CUT&RUNTools 2.0 (Yu et al., 2021)

No Signal

• A. Potential cause
  • a) Antibody error: The antibody that couldn't recognize the target antigen was used, which resulted in the subsequent pA-Tn5 being bound to the correct position, and the key steps of library construction could not be started; finally, no signal was generated.
  • b) Over-permeability: If the permeability condition is too intense, the nucleus will rupture, and the target protein will be lost with the nuclear content, which makes it impossible to detect the target signal in the experiment.
  • c) Failure of PCR amplification: During PCR amplification, unreasonable primer design, insufficient enzyme activity, or improper cycle number setting may lead to the failure of effective library construction, and finally, no signal can be detected.
• B. Solution strategy
  • a) Confirm the applicability of the antibody: Recheck the specificity and applicability of the antibody, and give priority to the antibody verified by CUT&Tag to ensure that the target antigen can be recognized.
  • b) Optimization of permeability conditions: Adjust the concentration of permeability reagent, treatment time, and other conditions to avoid excessive permeability and protect the nuclear structure and target protein.

To ensure the effectiveness of PCR amplification, carefully check the quality and activity of primers and enzymes for PCR amplification, and set the cycle number reasonably. qPCR can be used to monitor the amplification process in real time, or a positive control can be set (such as using samples of positive control antibodies for amplification) to ensure the success of library construction.

Similar performance using pA/MNase and pAG/MNase (Meers et al., 2019)

Conclusion

The efficient application of CUT&Tag technology depends on full preparation before the experiment, fine optimization of each step, and quick troubleshooting of common problems. Strict antibody verification, permeability optimization of adaptive cell types, perfect control settings, and parameter adjustment of labeling and library construction can ensure the success of the experiment to the greatest extent.

At the same time, the troubleshooting strategy, which aims at the common problems such as low library yield, high background noise, and no signal, can quickly locate and solve the obstacles in the experiment and provide reliable technical support for epigenetics research. With the continuous development of technology, CUT&Tag is expected to play a more important role in the frontier fields such as single-cell epigenetics and spatial epigenetics, and experimental optimization, and the accumulation of troubleshooting knowledge will lay a solid foundation for these frontier applications.

It is worth mentioning that professional reagents and technical support are crucial to the success of the CUT&Tag experiment. CD Genomics provides High quality service and professional team, which provides a strong guarantee for researchers to explore in the frontier field of epigenetics.

References

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2. 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.
3. Xu W, Ye Y, Sharrocks AD, Zhang W, Chen X. "Genome-wide Interrogation of Protein-DNA Interactions in Mammalian Cells Using ChIPmentation." STAR Protoc. 2020 1(3): 100187.
4. Yu F, Sankaran VG, Yuan GC. "CUT&RUNTools 2.0: a pipeline for single-cell and bulk-level CUT&RUN and CUT&Tag data analysis." Bioinformatics. 2021 38(1): 252-254.
5. Meers MP, Bryson TD, Henikoff JG, Henikoff S. "Improved CUT&RUN chromatin profiling tools." Elife. 2019 8:e46314.