Abstract
BackgroundOur understanding of eukaryotic gene regulation is limited by the complexity of protein–DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted nuclease cleavage method that profiles DNA-binding proteins, histones and chromatin-modifying proteins in situ with exceptional sensitivity and resolution.ResultsHere, we describe an automated CUT&RUN platform and apply it to characterize the chromatin landscapes of human cells. We find that automated CUT&RUN profiles of histone modifications crisply demarcate active and repressed chromatin regions, and we develop a continuous metric to identify cell-type-specific promoter and enhancer activities. We test the ability of automated CUT&RUN to profile frozen tumor samples and find that our method readily distinguishes two pediatric glioma xenografts by their subtype-specific gene expression programs.ConclusionsThe easy, cost-effective workflow makes automated CUT&RUN an attractive tool for high-throughput characterization of cell types and patient samples.
Highlights
Our understanding of eukaryotic gene regulation is limited by the complexity of protein–DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions
We modify the CUT&RUN protocol to profile chromatin proteins and modifications in a 96-well format on a liquid handling robot, beginning with permeabilized cells and ending with barcoded libraries that are ready to be pooled for sequencing. By applying this method to the H1 human embryonic stem cell line and the K562 leukemia cell line, we demonstrate that AutoCUT&RUN can be used to identify cell-type-specific promoter and enhancer activities, providing a means to quantitatively distinguish cell-types based on their unique gene regulatory programs
End-polishing and adapter ligation are performed on a separate thermocycler, and deproteinated CUT&RUN libraries are purified on the Biomek using Ampure XP magnetic beads both before and after PCR enrichment
Summary
Our understanding of eukaryotic gene regulation is limited by the complexity of protein–DNA interactions that comprise the chromatin landscape and by inefficient methods for characterizing these interactions. We recently introduced CUT&RUN, an antibody-targeted nuclease cleavage method that profiles DNA-binding proteins, histones and chromatin-modifying proteins in situ with exceptional sensitivity and resolution. Cells establish their distinct identities by altering activity of the cis-regulatory DNA elements that control gene expression [1, 2]. Defects in the nuclear proteins that recognize these cis-regulatory elements underlie many human diseases that often manifest in specific tissues and cell types [3,4,5,6,7]. We are only just beginning to appreciate how assessing the activity of cis-regulatory elements may be used in clinical settings for patient diagnosis [8]. Chromatin immunoprecipitation with antigen-specific antibodies combined with massively parallel sequencing (ChIP-seq) has been used extensively for epigenome profiling, but this method is labor-intensive, prone to artifacts [12,13,14], and requires high sequencing depth to distinguish weak signals from
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