TITLE: No Title
AUTHORS: 

[DESCRIPTION]
<p>Cleavage Under Targets and Release Using Nuclease (CUT&amp;RUN) is an epigenomic profiling strategy in which antibody-targeted controlled cleavage by micrococcal nuclease releases specific protein-DNA complexes into the supernatant for paired-end DNA sequencing. As only the targeted fragments enter into solution, and the vast majority of DNA is left behind, CUT&amp;RUN has exceptionally low background levels. CUT&amp;RUN outperforms the most widely used Chromatin Immunoprecipitation (ChIP) protocols in resolution, signal-to-noise, and depth of sequencing required. In contrast to ChIP, CUT&amp;RUN is free of solubility and DNA accessibility artifacts and can be used to profile insoluble chromatin and to detect long-range 3D contacts without cross-linking. Here we present an improved CUT&amp;RUN protocol that does not require isolation of nuclei and provides high-quality data starting with only 100 cells for a histone modification and 1000 cells for a transcription factor. From cells to purified DNA CUT&amp;RUN requires less than a day at the lab bench.</p>
<p> </p>
<p>In summary, CUT&amp;RUN has several advantages over ChIP-seq: (1) The method is performed in situ in non-crosslinked cells and does not require chromatin fragmentation or solubilization; (2) The intrinsically low background allows low sequence depth and identification of low signal genomic features invisible to ChIP; (3) The simple procedure can be completed within a day and is suitable for robotic automation; (4) The method can be used with low cell numbers compared to existing methodologies; (5) A simple spike-in strategy can be used for accurate quantitation of protein-DNA interactions. As such, CUT&amp;RUN represents an attractive replacement for ChIPseq, which is one of the most popular methods in biological research.</p>

[BEFORE_START]
<p><strong>REAGENT SETUP</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>5% Digitonin</strong> To reconstitute enough digitonin for an experiment, weigh out the powder in a 2 ml microcentrifuge tube, boil water in a small beaker in a microwave oven, and pipette in and out to warm the 1000 μL pipette tip. Pipette the hot water into the tube with the digitonin powder to make 5% (w/v), close the cap and quickly vortex on full until the digitonin is completely dissolved. If refrigerated, this stock can be used within a week, but will need reheating as the digitonin slowly precipitates. The effectiveness of digitonin varies between batches, so testing permeability of Trypan blue is recommended to determine the concentration to use for a cell type. We have obtained excellent results for K562 cells with 0.02-0.1% digitonin.</p>
<ul style="text-align: justify;">
<li><strong>CAUTION</strong>: Digitonin is toxic and care should be taken especially when weighing out the powder. A digitonin stock may be prepared by dissolving in dimethylsulfoxide (DMSO), but be aware that DMSO can absorb through the skin.</li>
</ul>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Binding buffer</strong> Mix 400 μL 1M HEPES-KOH pH 7.9, 200 μL 1M KCl, 20 μL 1M CaCl<sub>2</sub> and 20 μL 1M MnCl<sub>2</sub>, and bring the final volume to 20 ml with dH<sub>2</sub>O. Store the buffer at 4 °C for 6 months.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Concanavalin A-coated beads</strong> Gently resuspend and withdraw enough of the slurry such that there will be 10 μL for each final sample and/or digestion time point. Transfer into 1.5 ml Binding buffer in a 2 ml tube. Place the tube on a magnet stand to clear (30 s to 2 min). Withdraw the liquid, and remove from the magnet stand. Add 1.5 ml Binding buffer, mix by inversion or gentle pipetting, remove liquid from the cap and side with a quick pulse on a microcentrifuge. Resuspend in a volume of Binding buffer equal to the volume of bead slurry (10 μL per final sample).</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Wash buffer</strong> Mix 1 ml 1 M HEPES pH 7.5, 1.5 ml 5 M NaCl, 12.5 μL 2 M Spermidine, bring the final volume to 50 ml withdH<sub>2</sub>O, and add 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Store the buffer at 4 °C for up to 1 week.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Dig-wash buffer</strong> Mix 160-800 μL 5% Digitonin with 40 ml Wash buffer. The effectiveness of digitonin varies between batches, so testing permeability of Trypan blue is recommended to determine the concentration to use. We have obtained excellent results for K562 cells with 0.02-0.1% digitonin. Store the buffer at 4 °C for up to 1 day.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>Antibody buffer</strong> Mix 8 μL 0.5 M EDTA with 2 ml Dig-wash buffer and place on ice. Divide into aliquots for each antibody and add antibody solution or serum to a final concentration of 1:100 for to the manufacturer’s recommended concentration for immunofluorescence.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>2XSTOP</strong> To 4.2 ml dH<sub>2</sub>O add 340 μl 5M NaCl, 200 μL 0.5M EDTA, 100 μL 0.2M EGTA, 20 μL 5% Digitonin, 25 μL RNase A, 125 μL 2 mg/ml glycogen and 2 pg/ml heterologous spike-in DNA. Store the buffer at 4 °C  for up to 1 week.</p>
<ul>
<li style="text-align: justify;"><strong>CRITICAL STEP</strong>: Heterologous spike-in DNA for calibration should be fragmented down to ~200 bp mean size, for example, an MNase-treated sample of mononucleosome-sized fragments. As we use the total number of mapped reads as a normalization factor only, very little spike-in DNA is needed. For example, addition of 1.5 pg results in 1,000- 10,000 mapped spike-in reads for 1-10 million mapped experimental reads (in inverse proportion).</li>
</ul>

[GUIDELINES]
<div>
<p><strong>The protocol workflow is as follows:</strong></p>
</div>
<div>
<p> </p>
<p><strong>Day 1, Cells to DNA</strong></p>
<ul>
<li>Binding cells to beads (Steps 1-9, 30 min) </li>
<li>Bind (primary) antibodies (Steps 10-15, 15 min–overnight, with longer incubations providing higher yields)</li>
<li>Bind secondary antibody (as required) (Steps 16-22, 15 min-1.5 hr)</li>
<li>Bind Protein A-MNase fusion protein (Steps 23-30, 15 min–1.5 hr)</li>
<li>Targeted digestion (Steps 31-36, 45 min)</li>
<li>Target chromatin release (Steps 37-38, 20 min)</li>
<li>Option A: Fast DNA extraction by spin column (Steps 39-46, 20 min_</li>
<li>Option B: Alternate DNA extraction (preferred for quantitative recovery of ≤80 bp fragments) (Steps 47-58, 1.5 hr)</li>
</ul>
<p> </p>
<p><strong>Days 2-4</strong></p>
<ul>
<li>Library preparation and sequencing (Steps 59-64, 2-4 days) </li>
</ul>
</div>
<div> </div>
<div><strong>Day 5</strong></div>
<div>
<ul>
<li>Data processing and analysis (Steps 65-66, variable timing) </li>
</ul>
<p> </p>
<p> </p>
</div>
<p style="text-align: center;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rzqfw26.png" width="435" height="474" data-src="https://s3.amazonaws.com/pr-journal/rzqfw26.png" data-ofn="Screen Shot 2018-01-05 at 6.40.27 PM.png" /></p>
<p style="text-align: center;"> </p>
<p><strong>EQUIPMENT</strong></p>
<p> </p>
<ul>
<li>Centrifuge Eppendorf 5810, swinging bucket</li>
<li>Centrifuge Eppendorf 5424, fixed angle rotor</li>
<li>Centrifuge Eppendorf 5415R, refrigerated fixed angle rotor</li>
<li>Macsimag magnetic separator (Miltenyi, cat. no. 130-092-168), which allows clean withdrawal of the liquid from the bottom of 1.7 and 2 ml microfuge tubes.</li>
<li>Vortex mixer (e.g., VWR Vortex Genie)</li>
<li>Micro-centrifuge (e.g., VWR Model V)</li>
<li>1.5-ml microcentrifuge tubes (Genesee, cat. no. 22-282)</li>
<li>2-ml microcentrifuge tubes (Axygen, cat. no. MCT-200-C)</li>
<li>Tube rotator (Labquake, Thermo Fisher)</li>
<li>Heater block with wells for 1.5-ml microcentrifuge tubes</li>
<li>Water baths (set to 37°C and 70 °C)</li>
<li>MaXtract phase-lock microcentrifuge tubes (Qiagen, cat. no. 139046)</li>
<li>Capillary electrophoresis instrument (e.g. Agilent Tapestation 4200)</li>
<li>Qubit Fluorometer (Life Technologies, cat. no. Q33216)</li>
</ul>
<p> </p>
<p><strong>INTRODUCTION</strong></p>
<p> </p>
<p><strong>Experimental Design</strong></p>
<p> </p>
<p style="text-align: justify;">The CUT&amp;RUN method for the in situ targeted cleavage and release of chromatin complexes is straightforward and can be completed in under a day using standard lab equipment. Here we provide a detailed protocol and also provide various options that might be used to tailor the protocol to specific situations. One of the strengths of CUT&amp;RUN is that the entire reaction is performed in situ, whereby the antibody and pA-MN are free to diffuse into the nucleus. The original protocol used nuclei prepared by a combination of hypotonic lysis and treatment of cells with Triton X-100. This has been successful with a number of cell lines, but we have recently adapted the protocol to use cells permeabilized by the non-ionic detergent digitonin, which has been successfully used in other in situ methods, including ChEC-seq<sup>23</sup> and ATAC-seq<sup>24</sup>. Digitonin partitions into membranes and extracts cholesterol. Membranes that lack cholesterol are minimally impacted by digitonin<sup>25,26</sup>. Nuclear envelopes are relatively devoid of cholesterol compared to plasma membranes. As such, treatment of cells with digitonin represents a robust method for permeabilizing cells without compromising nuclear integrity<sup>26</sup>. The protocol described here uses digitonin, but it is possible that individual experimental situations call for generating intact nuclei by other means, and such nuclei can be prepared by a suitable method, bound to concanavalin A-coated beads as per our previously published work and then enter the protocol below at step 10<sup>20</sup>.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">One of the limitations of a protocol that has inherently low background and is amenable to low cell numbers is that the amount of DNA recovered can be very low, such that analysis even by sensitive capillary electrophoresis or picogreen assays (e.g. Agilent Tapestation and Qubit) are problematic. In addition, high resolution mapping techniques that cleave a minimal footprint are not suitable to PCR-based analysis of known binding loci, as it is not commonly possible to design ~50 bp PCR amplicons. As such, we recommend using a positive control antibody that</p>
<p style="text-align: justify;">targets an abundant epitope and therefore the DNA can be readily detected. We have successfully used a rabbit monoclonal antibody raised against H3K27me3, with capillary electrophoresis showing with the amount of cleaved fragments being proportional to the number of starting cells. A nucleosomal ladder is expected by Tapestation or other sensitiveelectrophoretic analysis method (<strong>Fig. 2</strong>), and the use of a monoclonal antibody avoids potential lot-to-lot variation that can complicate troubleshooting. For less abundant epitopes such as CTCF, it is harder to detect the cleaved fragments by even sensitive electrophoretic analysis (<strong>Supplementary Figure 1</strong>). Once the expected digested DNA pattern is observed for the positive control by capillary electrophoresis such as H3K27me3, it is not necessary to sequence this sample. As a negative control, we recommend the use of a non-specific rabbit IgG antibody that will randomly coat the chromatin at low efficiency without sequence bias. We do not recommend a no-antibody control, as the lack of tethering increases the possibility that slight carry-over of pA-MN will result in preferential fragmentation of hyper-accessible DNA.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rzvfw26.png" width="528" height="281" data-src="https://s3.amazonaws.com/pr-journal/rzufw26.png" data-ofn="Screen Shot 2018-01-05 at 6.46.41 PM.png" /></p>
<p style="text-align: center;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rzwfw26.png" width="251" height="314" data-src="https://s3.amazonaws.com/pr-journal/rzwfw26.png" data-ofn="Screen Shot 2018-01-05 at 6.47.07 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">In our previously published study, we showed that targeted cleavage occurred within seconds of adding Ca2+ ions, and by virtue of being a sterically regulated tethered reaction, the cleavage pattern was constant over time. However, longer digestion times release more material with no apparent change in the signal-to-nois ratio (<strong>Supplementary Figure 2</strong>). We therefore recommend digesting for 30 minutes as a starting point that can be tailored based upon epitope abundance and antibody concentration.</p>
<p> </p>
<p style="text-align: justify;"> </p>
<p><strong>Limitations</strong></p>
<p> </p>
<p style="text-align: justify;">As is the case with ChIP, the success of CUT&amp;RUN depends in large part on the affinity of the antibody for its target and its specificity under the conditions used for binding. Because antibodies bind to their epitopes in the solid state using CUT&amp;RUN, we would expect that antibodies successfully tested for specificity by immunofluorescence (IF) would be likely to work with CUT&amp;RUN, with the caveat that IF generally involves fixation, whereas formaldehyde fixation decreases the efficiency of CUT&amp;RUN.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">In the standard CUT&amp;RUN protocol we recommend allowing the cleaved chromatin complexes to diffuse out of the nuclei, thereby permitting simple isolation of the cut DNA from the supernatant fraction with the undigested genome retained in the intact nuclei. However, it is possible that a chromatin complex is too large to diffuse out or that protein-protein interactions retain the cleaved complex. In such cases, total DNA may be extracted after the digestion. By doing a very simple size selection using ½ volume of paramagnetic carboxylated beads (e.g. Agencourt AMPure XP beads) fragments below ~700 bp will be selected for. We previously showed that this strategy was successful for the ~1 MDa yeast RSC complex<sup>20</sup>.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>TROUBLESHOOTING</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><img id="s-mce-img" class="s-mce-img" src="https://s3.amazonaws.com/pr-journal/rzzfw26.png" width="539" height="401" data-src="https://s3.amazonaws.com/pr-journal/rzzfw26.png" data-ofn="Screen Shot 2018-01-05 at 8.57.32 PM.png" /></p>
<p style="text-align: justify;"> </p>
<p> </p>
<p style="text-align: justify;"><strong>AUTHOR CONTRIBUTIONS</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">P.S. and S.H developed the protocol, performed the experiments, analyzed the data and wrote the manuscript.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>ACKNOWLEDGEMENTS</strong></p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;">We thank Paul Talbert for helpful comments on the manuscript, Christine Codomo for preparing Illumina sequencing libraries and Jorja Henikoff for bioinformatics.</p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"> </p>
<p style="text-align: justify;"><strong>For additional information, comparison with other methods, and references please see the  full <a href="https://www.biorxiv.org/content/biorxiv/early/2017/09/24/193219.full.pdf" target="_blank">manuscript</a>.</strong></p>

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