Validation of Antibodies against Histone Modifications and Transcription Factors for ChIC/CUT&RUN Assays

Abstract

DNA‐protein interactions in the nuclei control a wide variety of biological processes such as development, differentiation, and cellular response to environmental changes. Chromatin immunoprecipitation (ChIP) has been used to analyze association of proteins with genomic DNA sequences. Although there have been several improvements with the technology over the decades, fundamental challenges remain with ChIP, including high background that limits sensitivity, high amounts of cellular input, and artifacts resulting from cross‐linking and solubilization.To overcome the challenges with ChIP, Cleavage Under Targets and Release Using Nuclease (CUT&RUN) was developed in Steven Henikoff’s lab by improving Chromatin Immune‐Cleavage (ChIC), which was originally developed in Ulrich Laemmli’s lab. In ChIC/CUT&RUN, binding sites of transcription factors in the genome were detected by targeting micrococcal nuclease conjugated with protein A (pA‐MN) through a specific antibody to detect genome‐wide transcription factor binding sites or histone modifications on native chromatin. Since the process doesn’t require cross‐linking and solubilization, and only the targeted fragments are released into solution and the majority of DNA is left behind, the results from ChIC/CUT&RUN assays have exceptionally low background levels compared with ChIP assays. As a result, ChIC/CUT&RUN provides high‐quality results using much smaller number of the cells (100 cells for a histone modification analysis and 1,000 cells for a transcription factor binding site analysis) than traditional ChIP assays.Here we performed ChIC/CUT&RUN assays with various antibodies against popular histone modifications including H3K4me3 and H3K27me3, and transcription factors such as CTCF. The sequencing data was compared with ChIP‐seq data in the ENCODE database. The sequencing data of ChIC/CUT&RUN assays with each of the antibodies tested showed very similar patters to the ENCODE ChIP‐seq data with the same targets. The results not only confirmed the power of the ChIC/CUT&RUN technology but also validated the suitability and comparability of these antibodies for use in a variety of studies.