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.
However it is not limited to profiling; CUT&RUN has the potential to replace all ChIP-based applications.
Discover all CUT&RUN Sets
|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||✔||✔||✔||✔|
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.
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.
- Hypotonic Lysis to release Nuclei
- Imobilize Nuclei with Magnetic Beads
- Incubate with Antibody against PoI
- Incubate with ProteinA-Mnase
- Add Ca+2 (Reaction Start)
- Add Chelator (Reaction Stop)
- Pellet oligonucleosome
Single Set Components available
- Guinea Pig anti-Rabbit IgG (Heavy & Light Chain) Antibody ABIN101961
- DYKDDDDK Tag antibody ABIN349610
- DYKDDDDK Tag antibody ABIN99294
- Rabbit anti-Mouse (Murine) IgG (Heavy & Light Chain) Antibody ABIN101776
- Histone Cluster 3, H3 (HIST3H3) (3meLys27) antibody ABIN2830935
- ConA Beads ABIN6923139
Suggested reagents based on Brahma et al.
- 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]
- Sandipan Brahma and Steven Henikoff (2018): "RSC-Associated Subnucleosomes Define MNase-Sensitive Promoters in Yeast". Mol Cell, Volume 73, Issue2, P238-249. [DOI]
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