Molecular Interaction Experiment: CUT& TAG targeted cutting and labeling

1、 Technical Introduction

CUT&TAG is a high-sensitivity epigenetic analysis technique based on antibody guided targeted chromatin cleavage and tagging. This technique overcomes the bottlenecks of traditional ChIP seq, such as low sensitivity, high background noise, and large sample demand. It can analyze chromatin features such as histone modifications and transcription factor binding at the single-cell level, becoming a new approach in epigenetic researchdirection.

2、 Technical principles

CUT&TAG achieves precise chromatin analysis through four key reactions:

1. Antibody targeted localization:Specific primary antibodies (such as H3K4me3 antibodies) bind to the target chromatin region, while Protein A/G secondary antibodies carry Tn5 transposase complexes (including sequencing adapters).

2. Targeted cutting and labeling:Tn5 transposase, guided by antibodies, performs DNA double strand cleavage and linker ligation on the target chromatin, with a cleavage range of approximately 50-300 bp on each side of the target site.

3. DNA fragment release:Protease K digests chromatin crosslinks and releases DNA fragments with adapters.

4. Library construction and sequencing:PCR amplification of target fragments, construction of Illumina sequencing library, sequencing depth:~20 million reads/sample (reduced by 50% compared to ChIP seq).

3、 Application scenarios

CUT&TAG reveals epigenetic differences among different subgroups in the tumor microenvironment at the single-cell level.

For example:

1. Triple negative breast cancer: Through single cell CUT&TAG, we found that FOXA1 regulated super enhancers were specifically activated in basal like subtypes, and these enhancers drove the expression of key genes of EMT (epithelial mesenchymal transformation) (such as SNAI1) and promoted metastasis.

2. Acute myeloid leukemia: Tracking bone marrow samples from recurrent patients, identifying dynamic changes in H3K4me1 modification in CD34+stem cells, which indicate the development of chemotherapy resistance and provide new targets for epigenetic targeted therapy.

3. Epigenetic reprogramming of embryonic development: CUT&TAG can track the dynamic establishment process of histone modifications during early embryonic development.

4. Mouse pre implantation embryos: H3K9me3 was found to begin depositing at the 2-cell stage, preferentially labeling retrotransposon regions (such as LINE-1), which maintain genomic stability by inhibiting transposon activity.

5. Human embryonic stem cells: Draw a dynamic map of H3K27ac in neural differentiation, locate the enhancer network regulated by SOX2 and OCT4, and reveal key nodes for pluripotency exit.

6. Regulation of immune cell function: Analyzing the epigenetic basis of T cell differentiation and functional status.

7. T cell depletion: In a chronic viral infection model, H3K27ac of exhausted CD8+T cells was found to be abnormally enriched at the PD-1 and TIM-3 loci, and the activity of these enhancers is directly regulated by TOX transcription factors.

8. Regulatory T cells (Treg): Combined with ATAC seq, FOXP3 stabilizes the inhibitory function of Treg cells by reshaping H3K4me3 modification, which relies on EZH2 mediated H3K27me3 synergistic inhibition.

4、 Technical advantages

1. High sensitivity (100 cell level detection): Tn5 transposase directly cleaves and connects sequencing adapters at the target site, avoiding random loss caused by DNA fragmentation (ultrasonic fragmentation) in ChIP seq. Compared with ChIP seq, CUT&TAG has an 8-fold increase in signal-to-noise ratio (S/N=12:1 vs 1.5:1) and a 40% increase in the number of detected differential peaks.

2. Single base resolution and precise localization: The anti-Tn5 complex only cleaves DNA near the binding site (± 50 bp), while ultrasound fragmentation of ChIP seq produces random fragments of 200-500 bp. In the analysis of p53 binding sites, CUT&TAG detected a single narrow peak (peak width ≈ 30 bp), while ChIP seq showed a broad peak (peak width ≈ 200 bp), which can accurately locate the TTGCCT motif center.

3. Compatibility between single-cell and spatial epigenomics: In combination with the 10x Genomics platform, single-cell H3K27ac atlas construction has been achieved by encapsulating single-cell and anti-Tn5 complexes in oil droplets through microfluidics (Nature Methods, 2022).

4. The newly developed Spatial CUT&Tag technology can locate the substructure distribution of H3K4me3 on tissue slices, such as visualizing hippocampal neuron activity related modifications in mouse brain slices.

5、 Sample type

Fresh cells (adherent cells/suspended cells), frozen cells, frozen tissues.

6、 Sample requirements

Cell samples: approximately 100000 (can be 1 well of a 24 well plate or stored in a centrifuge tube as suspended cells);

Frozen tissue: approximately 10-20mg.

7、 Sample preparation&transportation

Live cell samples: Fresh samples can be provided in close proximity, and fresh cell samples cultured in dishes/centrifuge tubes can be transported by express delivery at room temperature (latest next day delivery) within the city, either by flash delivery or personal delivery; For cells that are far away, they can be frozen at -80 ℃ for dry ice transportation;

Organizational sample: Frozen dry ice transportation at -80 ℃.

8、 Required reagents

ChIP grade antibody (IF grade antibody can also be selected when not available)

9、 Experimental cycle

1-2 weeks (excluding sequencing)

10、 Other

Subsequent testing: Real time PCR/sequencing