This work was financially supported in part by grants from the National Natural Science Foundation of China (NSFC, 31900395, 31971985, 31901430), and Fundamental Research Funds for the Central Universities, Hainan Yazhou Bay Seed Lab (JBGS, B21HJ0403), Hainan Provincial Natural Science Foundation of China (320LH002), and JCIC-MCP project.

1. Prepare transposase and stock solutions (Day 1)
NOTE: In this part, the oligonucleotide adapters are complexed with Tn5 transposase to make active transposase.
<ol>
	<li>Dilute primers (primer A, primer B, and primer C) to 100 &#181;M concentration using the annealing buffer (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA) (refer to Table 1 for sequence information of primers and Table 2 for the recipes for working solutions). Store at -20 &#17...

<ol>
	<li>Abascal, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Perspectives+on+ENCODE.">Perspectives on ENCODE.</a> Nature. 583 (7818), 693-698 (2020).</li><li>Park, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ChIP-seq:+advantages+and+challenges+of+a+maturing+technology.">ChIP-seq: advantages and challenges of a maturing technology.</a> Nature Reviews Genetics. 10 (10), 669-680 (2009).</li><li>Kaya-Okur, H. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=CUT&amp;Tag+for+efficient+epigenomic+profiling+of+small+samples+and+single+cells.">CUT&amp;Tag for efficient epigenomic profiling of small samples and single cells.</a> Nature Communications. 10 (1), 1930 (2019).</li><li>Tao, X., Feng, S., Zhao, T., Guan, X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+chromatin+profiling+of+H3K4me3+modification+in+cotton+using+CUT&amp;Tag.">Efficient chromatin profiling of H3K4me3 modification in cotton using CUT&amp;Tag.</a> Plant Methods. 16, 120 (2020).</li><li>Kaya-Okur, H. S., Janssens, D. H., Henikoff, J. G., Ahmad, K., Henikoff, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Efficient+low-cost+chromatin+profiling+with+CUT&amp;Tag.">Efficient low-cost chromatin profiling with CUT&amp;Tag.</a> Nature Protocols. 15 (10), 3264-3283 (2020).</li><li>Bartosovic, M., Kabbe, M., Castelo-Branco, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Single-cell+CUT&amp;Tag+profiles+histone+modifications+and+transcription+factors+in+complex+tissues.">Single-cell CUT&amp;Tag profiles histone modifications and transcription factors in complex tissues.</a> Nature Biotechnology. , (2021).</li><li>Paterson, A. H., Brubaker, C. L., Wendel, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+method+for+extraction+of+cotton+(Gossypium+spp.)+genomic+DNA+suitable+for+RFLP+or+PCR+analysis.">A rapid method for extraction of cotton (Gossypium spp.) genomic DNA suitable for RFLP or PCR analysis.</a> Plant Molecular Biology Reporter. 11 (2), 122-127 (1993).</li><li>Haring, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatin+immunoprecipitation:+optimization,+quantitative+analysis+and+data+normalization.">Chromatin immunoprecipitation: optimization, quantitative analysis and data normalization.</a> Plant Methods. 3 (1), 11 (2007).</li><li>Ouyang, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+low-input+profiling+of+histone+marks+in+plants+using+nucleus+CUT&amp;Tag.">Rapid and low-input profiling of histone marks in plants using nucleus CUT&amp;Tag.</a> Frontiers in Plant Science. 12, 634679 (2021).</li><li>Swift, J., Coruzzi, G. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+matter+of+time+-+How+transient+transcription+factor+interactions+create+dynamic+gene+regulatory+networks.">A matter of time - How transient transcription factor interactions create dynamic gene regulatory networks.</a> Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms. 1860 (1), 75-83 (2017).</li><li>Buenrostro, J. D., Wu, B., Chang, H. Y., Greenleaf, W. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ATAC-seq:+a+method+for+assaying+chromatin+accessibility+genome-wide.">ATAC-seq: a method for assaying chromatin accessibility genome-wide.</a> Current Protocols in Molecular Biology. 109, 21-29 (2015).</li></ol>

Here, we have described CUT&#38;Tag, a technology for generating DNA libraries at high resolution and exceptionally low background using a small number of cells with a simplified procedure compared to chromatin immunoprecipitation (ChIP). Our success with H3K4me3 profiling in cotton leaves suggests that CUT&#38;Tag, which was first designed for animal cells, can also be used for plant cells. Both the Tris buffer system commonly used for ChIP assay8 and the HEPES buffer system used for animal CUT&#...

Transcription factor binding DNA sites and open chromatin associated with histone modification marks serve critical functional roles in regulating gene expression and are the major focuses of epigenetic research1. Conventionally, chromatin immunoprecipitation assay (ChIP) coupled with deep sequencing (ChIP-seq) have been used for the genome-wide identification of specific chromatin histone modification or DNA targets with specific proteins, and is widely adopted in the field of epigenetics2. Cleavage under targets and tagmentation (CUT&amp;Tag) technology was originally developed by the Henikoff Lab to capture the protein-affiliated DNA fragments throughout the genome5. When compared to ChIP, CUT&amp;Tagcan generate DNA libraries at high resolution and exceptionally low background using a small number of cells with a simplified procedure3. To date, methods for the analysis of chromatin regions with specific histone modifications using CUT&amp;Tag have been established in animal cells4,5. Specifically, single-cell CUT&amp;Tag (scCUT&amp;Tag) has also been successfully developed for human tissues and cells6. However, due to the complexity of the cell wall and secondary metabolites, CUT&amp;Tag is still technically challenging for plant tissues. 
Previously, we reported a CUT&amp;Tag protocol using intact nuclei isolated from allotetraploid cotton leaves4. To demonstrate the efficiency of nuclei isolation and DNA capture using plant tissue, the profiling procedure is presented here. The major steps include intact nuclei isolation, in situ incubation with the antibody for chromatin modification, transposase incubation, adaptor integration, and DNA library preparation. The troubleshooting focuses on the CUT&amp;Tag library preparation for the plant nuclei isolation and quality control. 
In this study, we present a refined CUT&amp;Tag strategy for plants. Not only do we provide a step-by-step protocol for H3K4me3 profiling in cotton leaves, but we also provide an optimized methodology for plant nuclei isolation, including the strategy for selecting the concentration of detergent for proper cell lysis and the strategy to filter the nuclei. Detailed steps with critical comments are also included in this updated protocol.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Antibody</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Anti-H3K4me3</td>
			<td>Millipore</td>
			<td>07-473</td>
			<td></td>
		</tr>
		<tr>
			<td>Normal rabbit IgG</td>
			<td>Millipore</td>
			<td>12-370</td>
			<td></td>
		</tr>
		<tr>
			<td>Chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td></td>
			<td></td>
			<td>Make 10 mg/ml BSA stock solution. Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>digitonin (~50% (TLC)</td>
			<td>Sigma-Aldrich</td>
			<td>D141</td>
			<td>Make 5% digitonin stock solution (200 mg digitonin [~50% purity] to 2 mL DMSO). Note: Sterilize using a 0.22- micron filter. Store at -20&#176;C</td>
		</tr>
		<tr>
			<td>dimethyl sulfoxide (DMSO)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>chloroform</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ethylenediaminetetraacetic acid (EDTA)</td>
			<td></td>
			<td></td>
			<td>Make 0.5 M EDTA (pH = 8.5) stock solution. Note: Making 100 mL of 0.5-M EDTA (pH = 8.5) requires approximately 2 g of sodium hydroxide (NaOH) pellets to adjust the pH</td>
		</tr>
		<tr>
			<td>ethanol</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GlycoBlue Coprecipitant (15 mg/mL)</td>
			<td>Invitrogen</td>
			<td>AM9516</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium chloride (MgCl2)</td>
			<td></td>
			<td></td>
			<td>Make 1 M MgCl2 stock solution</td>
		</tr>
		<tr>
			<td>protease inhibitor cocktail</td>
			<td>Calbiochem</td>
			<td>539133-1SET</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride (KCl)</td>
			<td></td>
			<td></td>
			<td>Make 1 M KCl stock solution</td>
		</tr>
		<tr>
			<td>phenol:chloroform:isoamyl alcohol (25:24:1,v:v:v)</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride (NaCl)</td>
			<td></td>
			<td></td>
			<td>Make 5 M NaCl stock solution</td>
		</tr>
		<tr>
			<td>spermidine</td>
			<td></td>
			<td></td>
			<td>Make 2 M spermidine stock solution, store at -20&#176;C.</td>
		</tr>
		<tr>
			<td>sodium dodecyl sulfate (SDS)</td>
			<td></td>
			<td></td>
			<td>Make 10% SDS stock solution. Note: Do not autoclave; sterilize using a 0.22-micron filter</td>
		</tr>
		<tr>
			<td>Tris base</td>
			<td></td>
			<td></td>
			<td>Make 1 M Tris (pH = 8.0) stock solution</td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td></td>
			<td></td>
			<td>Make 20% Triton X-100 stock solution</td>
		</tr>
		<tr>
			<td>Enzyme</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Hyperactive pG-Tn5/pA-Tn5 transposase for CUT&#38;Tag</td>
			<td>Vazyme</td>
			<td>S602/S603</td>
			<td>Check the antibody affinity of the protein A or protein G that is fused with the Tn5. Generally speaking, proteins A and G have broad antibody affinity. However, protein A has a relatively higher affinity to rabbit antibodies and protein G has a relatively higher affinity to mouse antibodies. Select the appropriate transposase products that match your antibody.</td>
		</tr>
		<tr>
			<td>TruePrep Amplify Enzyme</td>
			<td>Vazyme</td>
			<td>TD601</td>
			<td></td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424R</td>
			<td></td>
		</tr>
		<tr>
			<td>PCR machine</td>
			<td>Applied Biosystems</td>
			<td>ABI9700</td>
			<td></td>
		</tr>
		<tr>
			<td>Orbital shaker</td>
			<td>MIULAB</td>
			<td>HS-25</td>
			<td></td>
		</tr>
		<tr>
			<td>NanoDrop One&#160; spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>ND-ONE-W</td>
			<td></td>
		</tr>
	</tbody>
</table>

profiling of h3k4me3 modification in plants using cleavage under targets and tagmentation

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&#38;Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&#38;Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

Figure 1&#160;depicts the CUT&#38;Tag workflow. Figure 2 shows the DAPI staining of the intact nuclei. The goal of the &#34;nuclei isolation&#34; step was to obtain the intact nuclei at a sufficient amount for the subsequent CUT&#38;Tag reaction. Figure 3 shows the agarose gel electrophoresis of PCR products. The IgG negative control is required in parallel when setting up the experiment. Compared with the IgG control group, the bul...

Watch this Scientific Journal Video about Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation at JoVE.com

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation

Cleavage under targets and tagmentation (CUT&amp;Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&amp;Tag strategy for the profiling of histone modifications in plants.

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their ...