An efficient targeted nuclease strategy for high-resolution mapping of DNA binding sites

Abstract

We describe Cleavage Under Targets and Release Using Nuclease (CUT&RUN), a chromatin profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. Unlike Chromatin Immunoprecipitation (ChIP), which fragments and solubilizes total chromatin, CUT&RUN is performed in situ, allowing for both quantitative high-resolution chromatin mapping and probing of the local chromatin environment. When applied to yeast and human nuclei, CUT&RUN yielded precise transcription factor profiles while avoiding crosslinking and solubilization issues. CUT&RUN is simple to perform and is inherently robust, with extremely low backgrounds requiring only ~1/10th the sequencing depth as ChIP, making CUT&RUN especially cost-effective for transcription factor and chromatin profiling. When used in conjunction with native ChIP-seq and applied to human CTCF, CUT&RUN mapped directional long range contact sites at high resolution. We conclude that in situ mapping of protein-DNA interactions by CUT&RUN is an attractive alternative to ChIP-seq.DOI: http://dx.doi.org/10.7554/eLife.21856.001

Unexpected conformational variations of the human centromeric chromatin complex

“…Of particular concern for centromere studies is the tendency of MNase, which is used for N-ChIP, to cause nibbling and internal cleavages (Brogaard et al 2012), leading to uncertainty as to whether particles are fully or partially wrapped (Hasson et al 2013). We recently introduced CUT&RUN, an efficient targeted nuclease method that is unrelated to ChIP in that it causes precise cleavage and release of intact antibody targeted particles without solubilizing the rest of the genome (Skene and Henikoff 2017b). In our most recent CUT&RUN protocol (Skene and Henikoff 2017a), antibodies are added to permeabilized cells bound to magnetic beads followed by addition of a protein fusion between MNase and protein A (pA-MN), which binds to the antibody.…”

“…When MNase is tethered to specific sites in CUT&RUN, there is no detectable nibbling, accessibility bias, or internal cleavages over a range of more than two orders of magnitude in digestion times even for highly AT-rich DNA. Moreover, because there is no chromatin solubilization, the CUT&RUN cleavage pattern of DNA extracted from the insoluble pellet can also be profiled (Skene and Henikoff 2017b). To adapt CUT&RUN for salt fractionation (CUT&RUN.Salt), chelation stop buffer was added without RNase, and, after removing the supernatant, we incubated the cell/bead pellet with 500 mM NaCl.…”

“…CUT&RUN of human K562 cells or nuclei was performed essentially as described (Skene and Henikoff 2017b) except that, after digestion, the protocol was modified to allow for salt fractionation, as described in the accompanying step-by-step protocol (Supplemental Material). Experiments shown in Figure 4 and Supplemental Table 1 used permeabilized cells rather than nuclei (Skene and Henikoff 2017a).…”

“…Merged pairs and paired-end 25-bp × 25-bp reads were mapped using Bowtie2 with following parameters: --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700. For CUT&RUN.Salt, read counts were calibrated using the spike-in control as described (Skene and Henikoff 2017b). Enrichment values represent the ratio of calibrated read counts for the specific antibody versus a nonspecific IgG control.…”

Remarkable Evolutionary Plasticity of Centromeric Chromatin

“…1, middle panel). More recently, we have used our novel CUT&RUN chromatin profiling method (Skene and Henikoff 2017) to show the presence of histone H2A in the CDEII particle (Fig. 2), definitively excluding the (Cse4/H4) 2 tetrasome model (Mizuguchi et al 2007;Wisniewski et al 2014).…”