What is CUT&Tag Sequencing? A Complete Beginner’s Guide

Traditional ChIP-seq relies on complex steps such as formaldehyde cross-linking and ultrasonic fragmentation, which suffer from bottlenecks including large sample requirements (millions of cells), long experimental cycles (5-7 days), and high background noise, limiting its application in rare samples (e.g., single-cell, clinical biopsy tissue) and low-abundance protein studies.

CUT&Tag (Cleavage Under Targets and Tagmentation) technology revolutionizes chromatin research by fusing antibody targeting with Tn5 transposase activity—it directly captures DNA fragments bound to target proteins without cross-linking, and simultaneously adds sequencing adapters, reducing sample requirements to 1000 cells or even single-cell levels, and shortening the experimental cycle to 1 day.

This article aims to systematically analyze the core principles, experimental procedures, technical advantages, and typical application scenarios (histone modification, transcription factor localization) of CUT&Tag for basic and clinical researchers, helping readers quickly grasp the underlying logic and practical value of this technology and providing methodological support for conducting high-resolution epigenetic research.

What is CUT&Tag sequencing?

CUT&Tag is an antibody-guided DNA-protein interaction research technology. It uses an engineered Tn5 transposase to precisely cleave the DNA region bound to the target protein and directly add sequencing adapters, enabling rapid construction of high-throughput sequencing libraries. Its core principle is: using antibodies to recognize specific proteins (such as histone modifiers or transcription factors), the Tn5 transposase complex carrying the sequencing adapter is targeted and bound to the target DNA region. Magnesium ions activate the transposase to cleave the DNA and insert the adapter, and finally, PCR amplification yields the sequencing library.

Compared to traditional ChIP-seq, CUT&Tag eliminates the need for formaldehyde cross-linking and ultrasonic fragmentation, shortening the experimental cycle to one day, and offering lower background noise and a higher signal-to-noise ratio.

In situ tethering for CUT&Tag chromatin profiling (Kaya-Okur HS et al., 2019)

CUT&Tag Technology Workflow

1. Sample Processing

• Nuclear Extraction: Live cells or nuclei are extracted using ConA magnetic beads, and nuclear membrane permeability is enhanced through digitonin permeation treatment.

• Antibody Incubation: The primary antibody specifically binds to the target protein (e.g., H3K27me3), and the secondary antibody bridges the Protein A/G-Tn5 transposase complex.

2. Transposation Reaction

• DNA Cutting and Adapter Addition: The Tn5 enzyme is activated in a Mg²⁺-containing buffer to cut the target DNA region and insert sequencing adapters.

• Fragment Purification: The DNA fragment is purified by digestion with proteinase K to remove antibodies and transposases.

3. Library Construction and Sequencing

• PCR Amplification: The purified DNA fragment is amplified, avoiding the end repair and ligation steps of traditional ChIP-seq.

• High-Throughput Sequencing: Paired-end sequencing is performed using the Illumina platform to obtain the sequence information of the target region.

Data Analysis Workflow

1. Quality Control and Alignment

• Quality Control Tools: FastQC checks sequencing quality, MultiQC integrates multi-indicator reports.

• Alignment Parameters: Use Bowtie2 to align to the reference genome, setting "local" and "very-sensitive" parameters to optimize alignment rate.

2. Peak Calling

• Tool Selection: MACS2 (recommended parameters: "nomodel", "shift", "75", "extsize", "150") or SEACR.

• Key Steps: Distinguish specific binding signals from background noise, and generate a "narrowPeak" file.

3. Functional Annotation and Visualization

• Gene Annotation: Use ChIPseeker or bedtools to associate peak positions with gene promoters, exons, and other regions.

• Visualization Tools: IGV displays peak distribution, and heatmaps analyze the binding strength of specific regions (such as promoters).

CUT&Tag vs. ChIP-seq: A Comparative Technical Analysis

Core Applications of CUT&Tag

1. Histone Modification Analysis

Detecting genome-wide histone modifications (e.g., H3K27me3, H3K4me1) to reveal epigenetic regulatory mechanisms of gene silencing or activation.

Case Study: Shi J et al. used CUT&Tag technology to map the high-resolution genome-wide distribution of four histone modifications (H3K4me1, H3K4me3, H3K27ac, H3K27me3) across seven embryonic developmental stages (from germinal bud to walnut stage). Analysis of chromatin state dynamics revealed:

• The dynamics of histone modifications (e.g., H3K4me3 enrichment in promoters, H3K27ac marking enhancers) were highly consistent with the timeline of transcriptome data, especially during ZGA (pre-blastocyst stage), where chromatin opening and transcriptional activation were synchronized (significantly increased gene expression during the blastocyst stage).

• This revealed the mechanism by which histone modification reprogramming (e.g., H3K27me3 established first, followed by upregulation of H3K4me1/3 later) and transcriptional activation synergistically drive development in L. vannamei embryonic development.

• The CUT&Tag technology has for the first time revealed a high-resolution dynamic landscape of histone modifications during L. vannamei embryonic development, revealing the synergistic regulatory mechanism between chromatin state transitions and gene expression, identifying key developmental genes and cis-regulatory elements, and providing the first epigenetic framework for crustacean embryo epigenomics, ZGA regulation, and aquaculture research.

Quality assessment of CUT&Tag at the nauplius III stage (Shi J et al., 2025)

2. Genome Regulation Research

This research reveals the distribution and dynamic changes of G4 and R-loop in the genome, providing new insights into non-classical genome structure and stress regulation.

Case Study: Li C et al., using G4-CUT&Tag and R-loop CUT&Tag technologies, discovered:

• Mapping the whole genome of endogenous G4 binding sites: 8195 G4 binding peaks were identified in HEK293T cells (G4 autobiotinylation method), with nearly 80% located in promoter regions and the remainder in intron/intergenic regions; these peaks highly overlapped with G4-CUT&Tag signals (72%) and were sensitive to G4 ligands (such as TMPyP4).

• Hemin treatment enhances G4 formation: 2 hours of Hemin treatment significantly increased the binding of Hemin to G4-CUT&Tag signals in promoters (N=5858) and enhancers (N=612), indicating that Hemin promotes G4 structure formation in specific regions.

• R-loop CUT&Tag results show that Hemin treatment reduces the R-loop signaling of Hemin binding to promoters and enhancers (reducing DNA:RNA hybrid structure).

Genome-wide profiling of hemin binding sites using biotin-PEG4-hemin and G4 self-biotinylation reaction (Li C et al., 2022)

3. Transcription Factor Binding Site Research

Precisely locating the DNA binding sites of transcription factors to elucidate their regulatory network on gene transcription.

Case Study: Li Y et al. used CUT&Tag technology to analyze the chromatin binding characteristics and regulatory mechanisms of LDB1 and its related factors. Key findings are as follows:

• LDB1 CUT&Tag analysis of six T-CEM cells identified 26,479 LDB1 binding peaks:
  • Approximately 40% of the peaks were located in promoter regions, and approximately 60% were located in enhancer-related regions (intergenic or intronic);
  • Modal analysis of these peaks showed that the LDB1 binding sites were rich in major transcription factor motifs associated with T-ALL progression (such as ETS1, RUNX1, GATA3, and MYB).
• The core transcriptional regulatory circuit constructed in T-ALL patient samples was validated using CUT&Tag (based on H3K27ac ChIP data);
• Co-localization of LDB1 with IRF1, ERG, and ETV6 on chromatin was observed, and these factors exhibited mutual transcriptional activation, indicating that LDB1 is a key maintainer of the core circuit. CUT & Tag analysis of other T-ALL cell lines (Loucy, Molt-4, J.gamma1) confirmed that the LDB1 complex binds to this enhancer, and that this enhancer is a key regulatory region for MYB transcription (MYB expression decreases and cell proliferation is inhibited after CRISPR interference).

Distribution of LDB1 binding to genomic regions in the 6 T-CEM cells, as assessed by CUT&Tag (Li Y et al., 2024)

4. Disease Mechanisms and Drug Screening

Analyzing the chromatin-binding characteristics of disease-related proteins to explore therapeutic targets.

Example: Zhao H et al., using CUT&Tag-seq (H2AZ1 antibody), RNA-seq, and ATAC-seq techniques, the relationship between H2AZ1 chromatin deposition patterns and the regulation of gene expression in lung cancer cells was revealed. Key findings are as follows:

• H2AZ1 binding peaks are highly enriched around transcription start sites (TSS) (approximately 40% in promoter regions ≤1kb, 60% in enhancer-related regions such as intergenic/intronic regions); binding is less frequent in gene body regions, and almost no binding occurs in the TSS "0 region" (RNA polymerase II/transcription factor binding region).

• Gene expression is significantly increased only when H2AZ1 binds to promoter regions ≤1kb or 5'UTR regions; gene expression is decreased/silenced when binding to gene bodies (promoter 1-3kb, introns, exons, etc.) or not at all.

• Gene expression was significantly higher when H2AZ1 deposition was combined with promoter region chromatin opening than when only H2AZ1 was bound or only chromatin was open. TCGA data validated this relationship as conserved in normal lung tissue and LUAD/LUSC.

• CUT&Tag technology clarified that H2AZ1 deposition around the TSS (especially in promoter regions ≤1kb) is key to active gene expression, and its synergistic effect with chromatin opening regulates gene expression. It also revealed that H2AZ1 participates in multiple cancer pathways and can bidirectionally regulate gene expression, providing new insights into the epigenetic mechanisms of lung cancer.

H2AZ1 binding to TSS causes higher gene expression and affects multiple cancer-related signaling pathways revealed by CUT&Tag-seq and RNA-seq (Zhao H et al., 2024)

Technical Limitations and Solutions

• Limitations:
  • Relies on high-quality antibodies; non-specific binding may affect results.
  • Limited resolution for very long DNA fragments (>1kb).
• Solutions:
  • Prioritize validated ChIP-grade antibodies.
  • Supplement long fragment information by combining with ATAC-seq or MNase-seq.

Conclusion

CUT&Tag, with its low starting amount, high sensitivity, and rapid workflow, has become an important tool in epigenetics research. Whether it's histone modification, transcription factor localization, or disease mechanism analysis, it provides high-resolution chromatin interaction maps. For beginners, it is recommended to start with the standard workflow and gradually master data analysis and result interpretation.

People Also Ask

What is CUT&Tag sequencing?

CUT&Tag-sequencing combines antibody-targeted controlled cleavage by a protein A-Tn5 fusion with massively parallel DNA sequencing to identify the binding sites of DNA-associated proteins. It can be used to map global DNA binding sites precisely for any protein of interest.

How many reads for CUT&Tag?

Libraries should be sequenced to a depth of 5-8 million total reads. For sufficient coverage, each library should generate 3-5 million unique reads (after removing multi-mapping reads, duplicate reads, reads in DAC exclusion list regions).

What is the difference between CUT&RUN and CUT&Tag?

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.

How does Tn5 tagmentation work?

Illumina developed the tagmentation protocol, in which a modified Tn5 enzyme cuts double-stranded DNA and concurrently ligates the linker sequences that are required for Illumina sequencing to both ends.

What is the sequencing depth of CUT&Tag?

CUT&Tag, the transposase only cuts chromatin at close proximity to the protein binding site, resulting in shorter lengths of DNA being sequenced. This allows lower sequencing depth (3-5 million reads) to generate robust data, with lower background signal than most ChIP-Seq assays.

References

1. Kaya-Okur HS, Wu SJ, Codomo CA, Pledger ES, Bryson TD, Henikoff JG, Ahmad K, Henikoff S. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nat Commun. 2019 Apr 29;10(1):1930.
2. Shi J, Qi Z, Yin M, Zeng Q, Hu J, Bao Z, Ye Z. Dynamic epigenomic landscape and gene regulatory networks during embryonic development in Pacific white shrimp (Litopenaeus vannamei) as revealed by histone modification profiling using CUT&Tag. Epigenetics Chromatin. 2025 Aug 4;18(1):50.
3. Li C, Yin Z, Xiao R, Huang B, Cui Y, Wang H, Xiang Y, Wang L, Lei L, Ye J, Li T, Zhong Y, Guo F, Xia Y, Fang P, Liang K. G-quadruplexes sense natural porphyrin metabolites for regulation of gene transcription and chromatin landscapes. Genome Biol. 2022 Dec 15;23(1):259.
4. Li Y, Zhang Z, Yu J, Yin H, Chu X, Cao H, Tao Y, Zhang Y, Li Z, Wu S, Hu Y, Zhu F, Gao J, Wang X, Zhou B, Jiao W, Wu Y, Yang Y, Chen Y, Zhuo R, Yang Y, Zhang F, Shi L, Hu Y, Pan J, Hu S. Enhancer looping protein LDB1 modulates MYB expression in T-ALL cell lines in vitro by cooperating with master transcription factors. J Exp Clin Cancer Res. 2024 Oct 9;43(1):283.
5. Zhao H, Wu X, Wang Y, Li X, Du Y, Zhou Z, Li Y, Liu Y, Zeng X, Chen G. Histone variant H2AZ1 drives lung cancer progression through the RELA-HIF1A-EGFR signaling pathway. Cell Commun Signal. 2024 Sep 26;22(1):453.