Improved Method to Study Protein-DNA Interactions

CUT&RUN

CUT&RUN, which stands for cleavage under targets and release using nuclease offers a new approach to pursue epigenetics1. CUT&RUN overcomes various downfalls of ChIP-Seq with improved workflow. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP (Fig 1), making CUT&RUN especially cost-effective for transcription factor and chromatin profiling1.

 Figure 1: Superior Peak Calling with CUT&RUN (green) in Comparison to ChIP-seq (orange)

However it is not limited to profiling; CUT&RUN has the potential to replace all ChIP-based applications.

Note: antibodies-online's CUT&RUN sets are now available! Discover the different compilations tailored to your needs Get started now!

Discover all CUT&RUN Sets

Table. 1: CUT&RUN Sets available. Contact our support for detailed information on each set.
Positive & Negative Control Rabbit anti-Mouse Secondary Rabbit anti-DYKDDDDK Primary Mouse anti-DYKDDDDK Primary ConA Beads
CUT&RUN Core ABIN6923134
CUT&RUN Core Sec ABIN6923133
CUT&RUN Core Direct ABIN6923132
CUT&RUN Core Complete ABIN6923131
CUT&RUN Pro ABIN6923138
CUT&RUN Pro Sec ABIN6923137
CUT&RUN Pro Direct ABIN6923136
CUT&RUN Pro Complete ABIN6923135
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Better Data
Only 1/3 of seq reads required
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Less Signal Noise
Easer Peak-Calling
Higher Reproducibility
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Less Sample
10x less sample in comparison to ChIP-seq
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Optimized Protocol
Obtain purified DNA from Cells within 1 day

CUT&RUN - Combining Perks of ChIP‐Seq Variations

As CUT&RUN is performed on intact cells or nuclei without fragmentation, it can be used to probe all genomic compartments. The cleaved chromatin complexes diffuse out of the nuclei where they can be harvested in supernatant. The rest of the undigested genome is retained in the intact nuclei (Fig 3). For ChIP-Seq on the other hand, the majority of chromatin is sheared or digested resulting in comparison in a worse signal-to-noise ratio (Fig 3-5). Consequently, ChIP-Seq requires more sequencing depth and with each sequencing run additional sample amount, labour time and money.

Extraction without fragmentation

The CUT&RUN is straightforward and can be completed in under a day using standard lab equipment. CUT&RUN is suitable for application down to 100 cells for profiling H3K27me3 or 1000 cells for CTCF sequence-specific DNA- binding protein 1. Therefore, CUT&RUN enables targeted genome-wide maps of protein- DNA interactions even for rare cell types. In comparison with XChiP-seq data (orange) both CUT&RUN runs (green) reveal reliable peaks with less background noise (Fig 1). One of the strengths of CUT&RUN is that the entire reaction is performed in situ.

In combination with a proximity ligation assay the generation of factor-specific high resolution maps of nuclear architecture is possible.

Experimental Design

Figure 2: CUT&RUN Diffusion

Fig.4.: Extraction without fragmentation.

  1. AB and Prot-A-MNase Fusion Protein diffuse in.
  2. Protein-DNA Complex diffuses into solution.

CUT&RUN

Figure 3: CUT&RUN

Native ChIP

Figure 4: Native ChIP (NChIP)

Cross-Linked ChIP

Figure 5: Cross-Linked ChIP

Importance of Antibody Selection

The successful execution of cleavage under target and release using nuclease hinges on antibody selection. A factor- or histone-specific antibody is bound to chromatin in situ followed by binding to the antibody of a protein A-micrococcal nuclease (pA-MNase) fusion. As is the case with ChIP, the success depends in large part on the affinity of the antibody for its target and its specificity under the conditions used for binding. The following list serves as an example, Brahma and Henikoff successfully utilized this compilation in 20182.

Note: You need help to choose the fitting antibody for your experiment? Our tech support will assist you!

Protocol Steps

  1. Hypotonic Lysis to release Nuclei
  2. Imobilize Nuclei with Magnetic Beads
  3. Incubate with Antibody against PoI
  4. Incubate with ProteinA-Mnase
  5. Add Ca+2 (Reaction Start)
  6. Add Chelator (Reaction Stop)
  7. Pellet oligonucleosome
  8. Sequencing

Single Set Components available

Suggested reagents based on Brahma et al.

Workflow

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References:

  1. Peter J. Skene and Steven Henikoff (2018): "CUT&RUN: Targeted in situ genome-wide profiling with high efficiency for low cell numbers”. Nature, Volume 13, pages 1006–1019. [PMID: 25652980]
  2. Sandipan Brahma and Steven Henikoff (2018): "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast". Mol Cell, Volume 73, Issue2, P238-249. [DOI]

KEYWORDS:
Cut and Run, CutAndRun, ChIP-seq, ChIP-sequencing, chromatin; chromosomes; epigenomics; gene expression; genetics; genomics; human; spike-in calibration; in situ profiling; transcription factors; A-Micrococcal Nuclease