Author response: Improved CUT&RUN chromatin profiling tools

摘要

Article Figures and data Abstract Introduction Results and discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Previously, we described a novel alternative to chromatin immunoprecipitation, CUT&RUN, in which unfixed permeabilized cells are incubated with antibody, followed by binding of a protein A-Micrococcal Nuclease (pA/MNase) fusion protein (Skene and Henikoff, 2017). Here we introduce three enhancements to CUT&RUN: A hybrid protein A-Protein G-MNase construct that expands antibody compatibility and simplifies purification, a modified digestion protocol that inhibits premature release of the nuclease-bound complex, and a calibration strategy based on carry-over of E. coli DNA introduced with the fusion protein. These new features, coupled with the previously described low-cost, high efficiency, high reproducibility and high-throughput capability of CUT&RUN make it the method of choice for routine epigenomic profiling. https://doi.org/10.7554/eLife.46314.001 Introduction Profiling the chromatin landscape for specific components is one of the most widely used methods in biology, and over the past decade, chromatin immunoprecipitation (ChIP) followed by sequencing (ChIP-seq) has become practically synonymous with genome-wide chromatin profiling (Landt et al., 2012; Schubert, 2018). However, the most widely used ChIP-seq protocols have limitations and are subject to artifacts (Jain et al., 2015; Park et al., 2013; Teves et al., 2016; Teytelman et al., 2013), of which only some have been addressed by methodological improvements (Brind'Amour et al., 2015; Kasinathan et al., 2014; Rhee and Pugh, 2011; Rossi et al., 2018; van Galen et al., 2016). An inherent limitation to ChIP is that solubilization of chromatin, whether by sonication or enzymatic digestion, results in sampling from the entire solubilized genome, and this requires very deep sequencing so that the sites of targeted protein binding can be resolved above background (Landt et al., 2012). To overcome this limitation, we introduced Cleavage Under Targets and Release Using Nuclease (CUT&RUN) (Skene and Henikoff, 2017), which is based on the chromatin immunocleavage (ChIC)-targeted nuclease strategy (Schmid et al., 2004): Successive incubation of unfixed cells or nuclei with an antibody and a Protein A-Micrococcal Nuclease (pA/MNase) fusion protein is followed by activation of MNase with calcium. In CUT&RUN, cells or nuclei remain intact throughout the procedure and only the targeted sites of binding are released into solution. Our CUT&RUN method dramatically reduced non-specific backgrounds, such that ~10 fold lower sequencing depth was required to obtain similar peak-calling performance (Skene and Henikoff, 2017). 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 (Skene et al., 2018). 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 (Janssens et al., 2018). Other advances based on our original CUT&RUN publication include CUT&RUN.Salt for fractionation of chromatin based on solubility (Thakur and Henikoff, 2018) and CUT&RUN.ChIP for profiling specific protein components within complexes released by CUT&RUN digestion (Brahma and Henikoff, 2019). CUT&RUN has also been adopted by others (Ahmad and Spens, 2018; Daneshvar et al., 2019; de Bock et al., 2018; Ernst et al., 2019; Federation et al., 2018; Hainer et al., 2019; Hainer and Fazzio, 2019; Hyle et al., 2019; Inoue et al., 2018; Liu et al., 2018; Menon et al., 2019; Oomen et al., 2019; Park et al., 2019; Roth et al., 2018; Uyehara and McKay, 2019; Zhang et al., 2019; Zheng and Gehri