High‐Resolution Chromatin Profiling Using CUT&RUN

Abstract

Determining the genomic location of DNA‐binding proteins is essential to understanding their function. Cleavage Under Targets and Release Using Nuclease (CUT&RUN) is a powerful method for mapping protein‐DNA interactions at high resolution. In CUT&RUN, a recombinant protein A–microccocal nuclease (pA‐MN) fusion is recruited by an antibody targeting the chromatin protein of interest; this can be done with either uncrosslinked or formaldehyde‐crosslinked cells. DNA fragments near sites of antibody binding are released from the insoluble bulk chromatin through endonucleolytic cleavage and used to build barcoded DNA‐sequencing libraries that can be sequenced in pools of at least 30. Therefore, CUT&RUN provides an alternative to ChIP‐seq approaches for mapping chromatin proteins, which typically have relatively high signal‐to‐noise ratios, while using fewer cells and at a lower cost. Here, we describe the methods for performing CUT&RUN, generating DNA‐sequencing libraries, and analyzing the resulting datasets. © 2019 by John Wiley & Sons, Inc.

Efficient generation of endogenous protein reporters for mouse preimplantation embryos

“…Importantly, the patterns of V5-tagged GATA3 and NANOG recapitulated their reported expression patterns in blastocysts (Home et al, 2009;Ralston et al, 2010;Strumpf et al, 2005). The expression pattern of CTCF has not been previously reported in blastocysts, although its DNA binding sites have been characterized in this context (Hainer and Fazzio, 2019). We detected tagged CTCF all nuclei of all cells of the blastocyst, consistent with RNA-seq data (Aksoy et al, 2013).…”

Dual threshold optimization and network inference reveal convergent evidence from TF binding locations and TF perturbation responses

“…One such technology is DamID, in which a DNA-methyltransferase is tethered to a DNA-binding protein and changes in DNA methylation relative to a control are assayed to determine binding location (van Steensel and Henikoff 2000;Hass et al 2015;Tosti et al 2018). Another is CUT&RUN, in which an endonuclease tethered to an antibody against a TF enters permeabilized nuclei and releases the DNA bound by the TF, which diffuses out of the cell and is recovered for sequencing (Skene and Henikoff 2017;Skene et al 2018;Hainer and Fazzio 2019;Meers et al 2019). A promising approach for measuring perturbation-response in mammalian cells is to transfect cells with a library of constructs encoding guide-RNAs that target a variety of TFs and then use single-cell RNA-seq to identify the TF perturbed and measure the response.…”

CUT&RUN detects distinct DNA footprints of RNA polymerase II near the transcription start sites

“…In this study, we have devised "CUT&RUN on plate" (CROP) for profiling protein-DNA interactions in adherent cells maintained in a multi-well cell culture plate. The current CUT&RUN protocol (Hainer and Fazzio 2019;Meers et al 2019a;Skene et al 2018) relies on Concanavalin A-coated magnetic beads to immobilise the cells before the targeted pAG-MNase cleavage. This immobilisation step introduces a range of shortcomings associated with the use of Concanavalin A-beads.…”