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URL: https://dx.doi.org/DOI:10.17504/protocols.io.bdwni7de

Authors: Antibodies Online Gmbh

Group: antibodies-online

Summary: CUT&RUN (Cleavage Under Targets and Release Using Nuclease) offers a novel approach to pursue epigenetics. The method is designed to map genome wide transcription factor binding sites, chromatin-associated complexes, and histone variants and post-translational modifications.Here we provide an updated CUT&RUN protocol that incorporates two variants, one is optimized to further reduce background noise which especially helps when working with low cell numbers and abundant antigens.In CUT&RUN is performed in situ on immobilized, intact cells without crosslinking. DNA fragmentation is achieved using micrococcal nuclease that is fused to Protein A and/or Protein G (pA/G-MNase). The fusion protein is directed to the desired target through binding of the Protein A/G moiety to the Fc region of an antibody bound to the target. DNA under the target is subsequently cleaved and released and the pA/G-MNase-antibody- chromatin complex is free to diffuse out of the cell. DNA cleavage products are extracted and then processed by next generation sequencing (NGS).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.

Affiliations: antibodies-online

External URL: https://campaigns.antibodies-online.com/cutandrun-sets/?utm_source=protocolsio&utm_medium=protocol&utm_content=cutrun

Version: 5

Publication Date: 2020

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