Improved CUT&RUN chromatin profiling tools

1Profiling 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. However, the most widely used ChIP-seq protocols have limitations and are subject to artifacts, of which only some have been addressed by methodological improvements. 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. To overcome this limitation, we introduced Cleavage Under Targets and Release Using Nuclease (CUT&RUN), which is based on the chromatin immunocleavage (ChIC)-targeted nuclease strategy: 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. 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. 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. Other advances based on our original CUT&RUN publication include CUT&RUN.Salt for fractionation of chromatin based on solubility and CUT&RUN.ChIP for profiling specific protein components within complexes released by CUT&RUN digestion. CUT&RUN has also been adopted by others, and since publication of our eLife paper we have distributed materials to >600 laboratories world-wide, with user questions and answers fielded interactively on our open-access Protocols.io site (dx.doi.org/10.17504/protocols.io.zcpf2vn).

2Broad implementation of CUT&RUN requires reagent standardization, and the rapid adoption of CUT&RUN by the larger community of researchers motivates the enhancements described here. First, the method requires a fusion protein that is not at this writing commercially available, and the published pA/MNase purification protocol is cumbersome, which effectively restricts dissemination of the method. Therefore, we have produced an improved construct with a 6-His-Tag that can be easily purified using a commercial kit, and by using a Protein A-Protein G hybrid, the fusion protein binds avidly to mouse antibodies, which bind only weakly to Protein A. Second, the original protocols are sensitive to digestion time, in that under-digestion results in low yield and over-digestion can result in pre-mature release of pA/MNase-bound complexes that can digest accessible DNA sites. To address this limitation, we have modified the protocol such that premature release is reduced, allowing digestion to near-completion for high yields with less background. Third, the current CUT&RUN protocol recommends a spike-in of heterologous DNA at the release step to compare samples in a series. Here we demonstrate that adding a spike-in is unnecessary, because the carry-over of E. coli DNA from purification of pA/MNase or pAG/MNase is sufficient to calibrate samples in a series.

3The pA/MNase fusion protein produced by the pK19-pA-MN plasmid requires purification from lysates of Escherichia coli overexpressing cells using an immunoglobulin G (IgG) column, and elution with low pH followed by neutralization has resulted in variations between batches. To improve the purification protocol, we added a 6-His tag into the pK19-pA-MN fusion protein (Figure 1A and Figure 1—figure supplement 1A). This allowed for simple and gentle purification on a nickel resin column (Figure 1—figure supplement 1B). In addition, we found that a commercial 6-His-cobalt resin kit also yielded pure highly active enzyme from a 20 ml culture, enough for ~10,000 reactions. Even when used in excess, there is no increase in release of background fragments (Figure 1—figure supplement 2), which indicates that the washes are effective in removing unbound fusion protein.

4In principle an epitope-tagged pAG/MNase could be used for chromatin pull-down from a CUT&RUN supernatant in sequential strategies like CUT&RUN.ChIP. However, in practice use of the 6-His tag is complicated by the requirement for a chelating agent to release the protein from the nickel resin. Therefore, we also added an HA (hemagglutinin) tag, which could be used to affinity-purify the complex of a directly bound chromatin particle with a primary antibody and the fusion protein.

5Protein A binds well to rabbit, goat, donkey and guinea pig IgG antibodies, but poorly to mouse IgG1, and so for most mouse antibodies, Protein G is generally used. To further improve the versatility of the MNase fusion protein, we encoded a single Protein G domain adjacent to the Protein A domain in the pK19-pA-MN plasmid. In addition, we mutated three residues in the Protein G coding sequence to further increase binding for rabbit antibodies. This resulted in a fusion protein that binds strongly to most commercial antibodies without requiring a secondary antibody. We found that for ordinary CUT&RUN applications pAG/MNase behaves very similarly to pA/MNase, but is more easily purified and is more versatile, for example allowing us to perform CUT&RUN without requiring a secondary antibody for mouse primary monoclonal antibodies (Figure 1B).

6When fragments are released by cleavage in the presence of Ca++ ions, the associated pA/MNase complex can digest accessible DNA. Although performing digestion at 0°C minimizes this artifact, eliminating premature release during digestion would allow for more complete release of target-specific fragments. Based on the observation that nucleosome core particles aggregate in high-divalent-cation and low-salt conditions, we wondered whether these conditions would prevent premature release of chromatin particles in situ. Therefore, we performed digestions in 10 mM CaCl2 and 3.5 mM HEPES pH 7.5. Under these high-calcium/low-salt conditions, chromatin is digested with no detectable release of fragments into the supernatant (Figure 2). 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. Furthermore, retention of the cleaved fragments within the nucleus under high-divalent cation/low-salt conditions could facilitate single-cell application of CUT&RUN.

7The high-calcium/low-salt protocol provided similar results using either pA/MNase and pAG/MNase (Figure 3). We also obtained similar results with either protocol for digestion time points over a ~ 30 fold range and for both supernatant and total DNA extraction (Figure 4—figure supplement 1). For antibodies to H3K27ac, libraries produced using the high-calcium/low-salt protocol showed improved consistency relative to the standard protocol when digested over an extended time-course (Figure 4), presumably because preventing release of particles during digestion avoids their premature release where they would artifactually digest accessible DNA. The close correlations between high-calcium/low-salt H3K27ac datasets for time points over a ~ 100 fold range occur with corresponding increases in the yield of fragments released into the supernatant during subsequent elution (Figure 4—figure supplement 2). This indicates that longer digestion times result in higher yields, with high signal-to-noise throughout the digestion series (Figure 4). Thus, this modification of CUT&RUN can reduce the risk of overdigestion for abundant epitopes such as H3K27ac, where premature release of pA-MNase-bound chromatin particles can increase background.

8We previously showed that CUT&RUN can be performed on insoluble protein complexes by extracting total DNA or by performing salt fractionation of the bead-bound cells and extracting DNA from the residual pellet. In either case, large DNA fragments were depleted using SPRI (AMPure XP) beads before library preparation. RNA polymerase II (RNAPII) from animal cells is insoluble when engaged, and requires harsh treatments for quantitative profiling using ChIP. To determine whether CUT&RUN can be used for insoluble chromatin complexes, we profiled Serine-5-phosphate on the C-terminal domain (CTD) of the Rpb1 subunit of RNAPII using both extraction of supernatant and of total DNA. This CTD phosphorylation is enriched in the initiating form of RNAPII, and we observed similar genic profiles for supernatant and total DNA (Figure 1B).

9Comparing samples in a series typically requires calibration for experimental quality and sequencing read depth. It is common to use background levels to calibrate ChIP-seq samples in a series and to define and compare peaks for peak-calling. However, the extremely low backgrounds of CUT&RUN led us to a calibration strategy based on spike-in of heterologous DNA, which has been generally recommended for all situations in which samples in a series are to be compared. In our current spike-in protocol, the heterologous DNA, which is typically DNA purified from an MNase digest of yeast Saccharomyces cerevisiae or Drosophila melanogaster chromatin, is added when stopping a reaction, and we adopted this spike-in procedure for the high-calcium/low-salt protocol described in the previous section. Interestingly, we noticed that mapping reads to both the spike-in genome and the E. coli genome resulted in almost perfect correlation (R2 = 0.97) between S. cerevisiae and E. coli in an experiment using pA/MNase in which the number of cells was varied over several orders of magnitude (Figure 5A). Near-perfect correlations (R2 = 0.96–0.99) between yeast spike-in and carry-over E. coli DNA were also seen in series using the same batch of pAG/MNase with high-calcium/low-salt digestion conditions (Figure 5B), and for both supernatant release and extraction and total DNA extraction (Figure 5C–D). These strong positive correlations are not accounted for by cross-mapping of the yeast spike-in to the E. coli genome, because omitting the spike-in for a low-abundance epitope resulted in very few yeast counts with high levels of E. coli counts (blue symbol in Figure 5C–D panels). As the source of E. coli DNA is carried over from purification of pA/MNase and pAG/MNase, the close correspondence provides confirmation of the accuracy of our heterologous spike-in procedure. Moreover, as carry-over E. coli DNA is introduced at an earlier step, and is cleaved to small mappable fragments that are released during digestion and elution, it provides a more desirable calibration standard than using heterologous DNA. High correlations were also seen between S. cerevisiae spike-in and E. coli carry-over DNA for pA-MNase in batches that we have distributed (Table 1). Therefore, data for nearly all CUT&RUN experiments performed thus far can be recalibrated post-hoc whether or not a spike-in calibration standard had been added.

10To explain the presence of carry-over E. coli DNA in proportion to the amount of yeast spike-in DNA, which is constant between samples in a series, we can exclude intracellular binding, because we observe proportionality between E. coli and yeast reads despite varying human cell numbers over two orders of magnitude (Figure 5A). Rather, we note that Concanavalin A binds to glycosylated immunoglobulins, and so the successive treatments of Con A bead-bound cells with excess antibody and Protein A(G)/MNase fusion protein will affix an amount of carry-over E. coli DNA to beads in proportion to the number of beads. Our use of a constant number of beads for all samples in a series to be compared would then have resulted in a constant amount of carry-over E. coli DNA. A similar inference of E. coli carry-over DNA suitable for calibration was noted for CUT&Tag, which suggests successive binding of antibodies and Protein A-Tn5 to the Con A beads used to immobilize cells. Thus, our calibration strategy might serve as a more general replacement for conventional spike-ins.

11Since its introduction in our original eLife paper, 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, for plant tissues and for high-throughput. The new CUT&RUN advances that we describe here are likely to be useful when applied in all of these protocols. Our improved CUT&RUN fusion construct simplifies reagent purification and eliminates the requirement for a secondary antibody against mouse primary antibodies. Our high-calcium/low-salt protocol minimizes time-dependent variability. Our discovery that carry-over E. coli DNA almost perfectly correlates with an added spike-in upgrades a contaminant to a resource that can be used as a spike-in calibration proxy, even post-hoc simply by counting reads mapping to the E. coli genome in existing CUT&RUN datasets.

12K562 cells were purchased from ATCC (#CCL-243) and cultured as previously described. All tested negative for mycoplasma contamination using MycoProbe kit.

13Hemagglutinin and 6-histidine tags were added to the carboxyl-terminus of pA-MNase using a commercially synthesized dsDNA fragment (gBlock) from Integrated DNA Technologies (IDT), which contains the coding sequence for both tags, glycine-rich flexible linkers and includes restriction sites for cloning. Another IDT gBlock containing the optimized protein-G coding sequence and homologous flanking regions to the site of insertion, was introduced via PCR overlap extension using Gibson Assembly Master Mix (New England Biolabs cat. #E2611), following the manufacturer’s instructions. The sequence-verified construct was transformed into JM101 cells (Agilent Technologies cat. #200234) for expression, cultured in NZCYM-Kanamycin (50 µg/ml) and induced with 2 mM Isopropyl β-D-1-thiogalactopyranoside following standard protein expression and purification protocols. The cell pellet was resuspended in 10 ml Lysis Buffer, consisting of 10 mM Tris-HCl pH 7.5, 300 mM NaCl, 10 mM Imidazole, 5 mM beta-mercaptoethanol, and EDTA-free protease inhibitor tablets at the recommended concentration (Sigma-Aldrich cat. #5056489001). Lysis using chicken egg white lysozyme (10 mg/mL solution, EMD cat. #71412 solution) was followed by sonication with a Branson Sonifier blunt-end adapter at output level 4, 45 s intervals for 5–10 rounds or until turbidity was reduced. The lysate was cleared by high-speed centrifugation and purified over a nickel-agarose column, taking advantage of the poly-histidine tag for efficient purification via immobilized metal affinity chromatography. Cleared lysate was applied to a 20 ml disposable gravity-flow column 1.5 ml (0.75 ml bed volume) of NI-NTA agarose (Qiagen cat. #30210), washed twice in three bed volumes of Lysis Buffer. Lysate was applied followed by two washes at five bed volumes of 10 mM Tris-HCl pH 7.5, 300 mM NaCl, 20 mM Imidazole, 0.03% ZWITTERGENT 3–10 Detergent (EMD Millipore cat. #693021) and EDTA-free protease inhibitor tablets. Elution was performed with 1 ml 10 mM Tris-HCl pH 7.5, 300 mM NaCl, 250 mM Imidazole and EDTA-free protease inhibitor tablets. Eluate was dialyzed twice against a 750 ml volume of 10 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM PMSF to remove imidazole. Glycerol was then added to 50%, aliquots stored at –80°C for long term storage and –20°C for working stocks.

14For purification, we used either the nickel-based protocol or the Pierce Cobalt kit (Pull-Down PolyHis Protein:Protein Interaction Kit cat. #21277 from Thermo Fisher). Similar results were obtained using either the nickel or cobalt protocol, although the cobalt kit alleviated the need for a sonicator, using a fifth of the starting material from either fresh culture or a cell pellet frozen in lysis buffer, and yielded more protein per volume of starting material. With the cobalt kit, 20 ml of culture yielded ~100 µg of fusion protein.

15Log-phase cultures of K562 cells were harvested, washed, and bound to activated Concanavalin A-coated magnetic beads, then permeabilized with Wash buffer (20 mM HEPES, pH7.5, 150 mM NaCl, 0.5 mM spermidine and a Roche complete tablet per 50 ml) containing 0.05% Digitonin (Dig-Wash) as described. The bead-cell slurry was incubated with antibody in a 50–100 µL volume for 2 hr at room temperature or at 4°C overnight on a nutator or rotator essentially as described. In some experiments, cells were permeabilized and antibody was added and incubated 2 hr to 3 days prior to addition of ConA beads with gentle vortexing; similar results were obtained (e.g. Figure 2B–D), although with lower yields. After 2–3 washes in 1 ml Dig-wash, beads were resuspended in 50–100 µL pA/MNase or pAG/MNase and incubated for 1 hr at room temperature. After two washes in Dig-wash, beads were resuspended in low-salt rinse buffer (20 mM HEPES, pH7.5, 0.5 mM spermidine, a Roche mini-complete tablet per 10 ml and 0.05% Digitonin). Tubes were chilled to 0°C, the liquid was removed on a magnet stand, and ice-cold calcium incubation buffer (3.5 mM HEPES pH 7.5, 10 mM CaCl2, 0.05% Digitonin) was added while gently vortexing. Tubes were replaced on ice during the incubation for times indicated in each experiment, and within 30 s of the end of the incubation period the tubes were replaced on the magnet, and upon clearing, the liquid was removed, followed by immediate addition of EGTA-STOP buffer (170 mM NaCl, 20 mM EGTA, 0.05% Digitonin, 20 µg/ml glycogen, 25 µg/ml RNase A, 2 pg/ml S. cerevisiae fragmented nucleosomal DNA). Beads were incubated at 37°C for 30 min, replaced on a magnet stand and the liquid was removed to a fresh tube and DNA was extracted as described. A detailed step-by-step protocol is available at https://www.protocols.io/view/cut-amp-run-targeted-in-situ-genome-wide-profiling-zcpf2vn. Extraction of pellet and total DNA was performed essentially as described.

16The size distribution of libraries was determined by Agilent 4200 TapeStation analysis, and libraries were mixed to achieve equal representation as desired aiming for a final concentration as recommended by the manufacturer. Paired-end Illumina sequencing was performed on the barcoded libraries following the manufacturer’s instructions. Paired-end reads were aligned using Bowtie2 version 2.2.5 with options: --local --very-sensitive-local --no-unal --no-mixed --no-discordant --phred33 -I 10 -X 700. For MACS2 peak calling, parameters used were macs2 callpeak – t input_file –p 1e-5 –f BEDPE/BED(Paired End vs. Single End sequencing data) –keep-dup all –n out_name. Some datasets showed contamination by sequences of undetermined origin consisting of the sequence (TA)n. To avoid cross-mapping, we searched blastn for TATATATATATATATATATATATAT against hg19, collapsed the overlapping hits into 34,832 regions and intersected with sequencing datasets, keeping only the fragments that did not overlap any of these regions.

17If digestion and fragment release into the supernatant occur linearly with time of digestion until all fragments within a population are released, then we expect that CUT&RUN features will be linearly correlated within a time-course series. For CTCF, features were significant CTCF motifs intersecting with DNAseI hypersensitive sites. For H3K27Ac and H3K4me2, we called peaks using MACS2 and calculated the Pearson correlation coefficients between time points, displayed as a matrix of R2 values, using the following procedure:

18Aligned fastq files to unmasked genomic sequence using Bowtie2 version 2.2.5 to UCSC hg19 with parameters: --end-to-end --very-sensitive --no-mixed --no-discordant -q --phred33 -I 10 -X 700.

19Extracted properly paired read fragments from the alignments and pooled fragments from multiple samples.

20Compared pooled fragments with (TA)n regions of hg19 and kept those fragments that did NOT overlap any (TA)n region using bedtools 2.21.0 with parameters: intersect -v -a fragments.bed -b TATA_regions.bed>fragments_not_TATA.bed.

21Found peaks using macs2 2.1.1.20160309 with parameters: callpeak -t fragments_not_mask.bed -f BED -g hs --keep-dup all -p 1e-5 -n not_mask –SPMR.

22Made scaled fractional count bedgraph files for each sample from bed files made in step 2. The value at each base pair is the fraction of counts times the size of hg19 so if the counts were uniformly distributed the value would be one at each bp.

23Extracted bedgraph values for ±150 bps around peak summits for IgG sample and computed their means, which resulted in one mean score per peak.

24Removed peaks from macs2 results in step four if the mean score was greater than the 99th percentile of all IgG scores to make a subset of the peaks lacking the most extreme outliers.

25Extracted bedgraph values for ±150 bps around the subset of peak summits from step seven for all samples and computed their means, which resulted in a matrix with columns corresponding to samples and one row per peak.

26Computed correlations of matrix in eight using R 3.2.2 cor(matrix, use=‘complete.obs’) command.