Beginner’s Guide: How to Start CUT&Tag Analysis
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High-purity protein A/G-Tn5 fusion enzymes for consistent and robust performance.
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A rigorously quality-controlled antibody library optimized for CUT&Tag applications.
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Comprehensive technical support to help users troubleshoot and optimize protocols.
Cleavage Under Targets and Tagmentation (CUT&Tag) is a chromatin profiling technique that has gained popularity due to its high sensitivity, low background noise, and operational simplicity. However, researchers new to this method may face challenges in getting started. This guide offers a comprehensive introduction for CUT&Tag beginners, covering experimental design, reagent selection, step-by-step protocols, and data analysis to facilitate the successful implementation of CUT&Tag experiments.
Defining Experimental Objectives and Design
Before initiating the experiment, it is essential to define the study objectives:
1. Chromatin target of interest (e.g., histone modification or transcription factor).
2. Sample type and number of cells (starting with 1,000–5,000 cells is recommended).
3. Selection of control group (e.g., IgG antibody or input DNA).
With these components clarified, detailed experimental planning can proceed.
Essential Reagents and Consumables
The success of CUT&Tag analysis largely depends on selecting suitable reagents and materials. The following are the basic requirements:
1. Key Reagents
(1) Primary antibody: High specificity is crucial; ChIP-grade antibodies are recommended.
(2) Protein A/G-Tn5 transposase: This core enzyme complex should be sourced from reputable suppliers, such as the high-efficiency fusion proteins provided by MtoZ Biolabs.
(3) Permeabilization and activation buffers: Commercial kits are recommended to enhance reproducibility and reduce procedural error.
2. Required Consumables
(1) Low-binding tubes.
(2) PCR tubes (compatible with qPCR and high-throughput sequencing).
(3) PCR kit containing high-fidelity DNA polymerase.
(4) DNA purification kit.
Detailed Protocol for CUT&Tag Experiments
The following outlines key procedural steps in CUT&Tag analysis to help new users implement the workflow effectively:
1. Cell Sample Preparation and Permeabilization
Collect the cells and wash them 1–2 times with PBS. Resuspend in permeabilization buffer and incubate for 10 minutes to allow membrane permeabilization and facilitate nuclear entry of antibodies.
2. Primary Antibody Incubation
Add the specific primary antibody and incubate at 4°C for 1–2 hours or overnight to ensure adequate binding to the target protein.
3. Incubation with Protein A/G-Tn5 Fusion Complex
Introduce the fusion enzyme complex and incubate at room temperature or 4°C for 1 hour to enable accurate targeting of chromatin regions.
4. Tagmentation (Chromatin Fragmentation and Labeling)
Add Mg²⁺ activation buffer and incubate at 37°C for 5–10 minutes. Controlled reaction time ensures effective and precise fragmentation while avoiding over-digestion.
5. DNA Release and PCR Amplification
Add proteinase K, followed by incubation at 50–55°C for 30 minutes and then 95°C for 10 minutes to release labeled DNA fragments. Amplify these fragments using PCR with high-fidelity DNA polymerase to minimize the introduction of sequencing artifacts or bias.
CUT&Tag Sequencing and Data Analysis
After completing CUT&Tag experiments, the resulting DNA fragments require high-throughput sequencing and analysis. The main steps include:
1. Sequencing Platform
High-throughput sequencing is typically performed using the Illumina platform. A sequencing depth of 10–20 million reads per sample is generally sufficient for most applications.
2. Quality Control
Use tools such as FastQC and MultiQC for assessing read quality.
3. Read Alignment and Peak Calling
Commonly used software includes:
(1) Bowtie2/BWA for alignment to the reference genome.
(2) MACS2 for peak identification.
4. Downstream Analysis and Visualization
Use IGV (Integrative Genomics Viewer) for visualizing peak signals and chromatin distribution. Employ statistical tools such as DESeq2 or EdgeR for analyzing epigenetic differences across conditions or cell populations.
Frequently Encountered Issues
1. High Background Signals
Reduce incubation time, especially during the tagmentation step. Use well-validated, high-quality antibodies to minimize non-specific binding.
2. Low Amplification Yield or Library Concentration
Increase the number of PCR cycles (10–15 cycles are typically sufficient). Verify the activity of the protein A/G-Tn5 enzyme and review procedural steps for accuracy.
To support researchers in efficiently executing CUT&Tag workflows, MtoZ Biolabs provides:
By following the procedures outlined above and utilizing high-quality reagents and professional support, even beginner researchers can effectively carry out the entire CUT&Tag workflow, from experimental design to data analysis. MtoZ Biolabs is committed to enabling accurate and reproducible CUT&Tag analysis, empowering researchers to embark on epigenetic research with confidence and precision.
MtoZ Biolabs, an integrated chromatography and mass spectrometry (MS) services provider.
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