2020/03/19 by Antibodies Online Gmbh · 1 citation
Biochemistry, Genetics and Molecular Biology · #RNA modifications and cancer #Advanced biosensing and bioanalysis techniques #Genomics and Chromatin Dynamics
paper · pdf · doi:10.17504/protocols.io.bdwni7de
openalex publication_date 2020/03/19 · openalex created_date 2025/10/10 · openalex updated_date 2026/07/14
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.