A fluorescence-based protocol to quantitatively titrate CUT&RUN buffer components

Abstract

Summary: Cleavage under targets & release using nuclease (CUT&RUN) is a technique for identifying genomic sites where proteins or histone modifications are present in chromatin in permeabilized cells. Here, we present a fluorescence-based protocol to quantitatively titrate CUT&RUN buffer components, for efficient cell permeabilization and retention of target epitopes on chromatin. We describe steps for capturing cells on concanavalin A beads and using a fluorescently labeled secondary antibody to titrate concentrations of digitonin and NaCl in CUT&RUN buffers. We then detail procedures for fluorescence imaging to identify optimal conditions.For complete details on the use and execution of this protocol, please refer to Lerner et al.1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

Keywords

• Cell Biology
• Cell-based Assays
• ChIP-seq
• Chromatin immunoprecipitation (ChIP)
• Genetics
• Molecular Biology