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Cleavage Under Targets and Tagmentation (CUT&Tag)

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The Introduction of CUT&Tag Sequencing

The advent of massively parallel sequencing and the dramatic reduction in cost per base has fueled a genomics revolution, however, the full promise of epigenomic profiling has lagged owing to limitations in methodologies used for mapping chromatin fragments to the genome. Chromatin immunoprecipitation with sequencing (ChIP-seq) has been an effective technical method to study the binding position of histone modifications and transcription factors on the genome. However, ChIP-seq suffers from low signals, high backgrounds and epitope masking due to cross-linking, and low yields require large numbers of cells. Currently, alternatives to ChIP include enzyme-tethering methods for unfixed cells, such as DamID, ChEC-seq, CUT&RUN(Cleavage under targets and release using nuclease), where a specific protein of interest is targeted in situ and then profiled in genome-wide scale, and CUT&Tag.

CUT&Tag, which is the abbreviation for Cleavage Under Targets and Tagmentation, is a molecular biology method that researchers use to investigate interactions between proteins and DNA and to identify DNA binding sites for their protein of interest.

In CUT&Tag, a chromatin protein is bound in situ by a specific antibody, which then tethers a protein A-Tn5 transposase fusion protein. Activation of the transposase results in the chromatin being cut close to the protein binding site and simultaneous addition of the NGS adapter DNA sequences, and efficiently generates fragment libraries with high resolution and exceptionally low background. All steps from live cells to sequencing-ready libraries can be performed in a single tube on the benchtop or a microwell in a high-throughput pipeline, and the entire procedure can be performed in one day.

The CUT&Tag method is very sensitive, it has been reported to work with as few as 60 cells for some histone modifications. With ChIP, sonication randomly shears chromatin and the antibody immunoprecipitates DNA with different lengths, usually several hundred base pairs in length. However, with 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. Finally, because the CUT&Tag protocol uses intact cells as the starting material, rather than sonicated chromatin, it can be adapted to single-cell experiments (scCUT&Tag).

The Introduction of CUT&Tag Sequencing

Key Features and Advantages

CUT&Tag is a newer molecular biology method for investigating protein-DNA interactions and offers some advantages over ChIP-Seq and CUT&RUN:

1. CUT&Tag is compatible with low cell numbers

In the first publication describing the CUT & Tag method, as low as 60 cells were sufficient to analyze H3K27me3 profiles across the genome.

2. CUT&Tag does not require fixation or sonication

CUT&Tag is performed on native (unfixed) cells or nuclei, avoiding the need for fixation, chromatin preparation, and sonication steps of standard ChIP workflows.

3. CUT&Tag is fast

4. Less sequencing is required for CUT&Tag

The shorter DNA sequences isolated by CUT&Tag means that it doesn’t have the same deep sequencing requirements as ChIP-Seq. 3-5 million reads are sufficient to generate robust data following CUT&Tag protocol, which is approximately 10-fold less sequencing than ChIP-Seq.

5. CUT&Tag is also more easily adaptable to single-cell analyses.

Project Workflow

The CUT&Tag protocol consists of 3 main steps:

  • Permeabilization of the native/unfixed cells
  • Incubation with the specific antibody
  • Targeted tagmentation.

The Introduction of CUT&Tag SequencingFig. 1 In situ tethering for CUT&Tag chromatin profiling. a. The steps in CUT&Tag. Added antibody (green) binds to the target chromatin protein (blue) between nucleosomes (gray ovals) in the genome, and the excess is washed away. A second antibody (orange) is added and enhances tethering of pA-Tn5 transposome (gray boxes) at antibody-bound sites. After washing away excess transposome, addition of Mg++ activates the transposome and integrates adapters (red) at chromatin protein binding sites. After DNA purification genomic fragments with adapters at both ends are enriched by PCR. b. CUT&Tag is performed on a solid support. Unfixed cells or nuclei (blue) are permeabilized and mixed with antibody to a target chromatin protein. After addition and binding of cells to Concanavilin Acoated magnetic beads (M), all further steps are performed in the same reaction tube with magnetic capture between washes and incubations, including pA-Tn5 tethering, integration, and DNA purification

Service Specifications

Sample Requirements

  • Sample species: Human, Rat, Mouse
  • Sample type: Cell, Tissue
  • Cells: > 60 cells
  • Tissue: 5mg
Sequencing Strategy

  • Because of the very low background with CUT&Tag, typically 3 million paired-end reads suffice for nucleosome modifications, even for the human genome.
  • Illumina PE150
Data Analysis

  • Data quality control
  • Genomic alignment analysis
  • Genomic enrichment analysis
  • Motif analysis
  • Super enhancer analysis
  • More data mining upon your request

The Introduction of CUT&Tag Sequencing

At CD Genomics, we provide you with high-quality sequencing and comprehensive bioinformatics analysis for your CUT&Tag project, enabling accurately screen and determine the binding position of histone modifications and transcription factors in the whole genome. If you have additional requirements or questions, please feel free to contact us.

Reference:

  1. Kaya-Okur HS, et al. CUT&Tag for efficient epigenomic profiling of small samples and single cells. Nature Communication, 2019.
* For Research Use Only. Not for use in diagnostic procedures.
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