Automated in situ chromatin profiling efficiently resolves cell types and gene regulatory programs

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.Electronic supplementary materialThe online version of this article (10.1186/s13072-018-0243-8) contains supplementary material, which is available to authorized users.

Improved CUT&RUN chromatin profiling and analysis tools

“…Since its introduction in our original eLife paper (1), the advantages of CUT&RUN over ChIP-seq has led to its rapid adoption, including publication of new CUT&RUN protocols for low cell numbers (14,23), for plant tissues (25) and for high-throughput (15). The new CUT&RUN advances that we describe here are likely to be useful when applied in all of these protocols.…”

“…We expect that as the value of precise fragment information becomes better appreciated, for example in inferring chromatin dynamics (47), our block aggregation strategy will become increasingly powerful. Cell culture K562 cells and H1 hESCs were cultured as previously described (15). Definitive endoderm (DE) was derived from a culture of H1 hESCs using the StemDiff Definitive Endoderm kit (StemCell Technologies).…”

“…Reactions are halted by transferring the tube to a magnet, removing the liquid, and adding elution buffer containing 150 mM NaCl, 20 mM EGTA and 25 µg/ml RNAse A, which releases the small DNA fragments into the supernatant. These conditions are compatible with direct end-polishing and ligation used for AutoCUT&RUN (15). Furthermore, retention of the cleaved fragments within the nucleus under high-divalent cation/low-salt conditions could facilitate single-cell application of CUT&RUN.…”

“…K562 cells and H1 hESCs were cultured as previously described (15). Definitive endoderm (DE) was derived from a culture of H1 hESCs using the StemDiff Definitive Endoderm kit (StemCell Technologies).…”

“…In addition, CUT&RUN provides near base-pair resolution, and our most recently published benchtop protocol is capable of profiling ~100 human cells for an abundant histone modification and ~1000 cells for a transcription factor (14). The simplicity of CUT&RUN has also resulted in a fully automated robotic version (AutoCUT&RUN) in which the high reproducibility and low cost makes it ideally suited for high-throughput epigenomic profiling of clinical samples (15). Other advances based on our original CUT&RUN publication include CUT&RUN.Salt for fractionation of chromatin based on solubility (16) and CUT&RUN.ChIP for profiling specific protein components within complexes released by CUT&RUN digestion (17).…”