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 &#176;C until use.</li>
	<li>Set up the following two reactions in PCR tubes: Reaction 1 (adaptor AB), 10 &#181;L of 100 &#181;M primer A, 10 &#181;L of 100 &#181;M primer B; Reaction 2 (adaptor AC), 10 &#181;L of 100 &#181;M primer A, 10 of &#181;L 100 &#181;M primer C.</li>
	<li>Anneal the adapters in the PCR machine using the following program: heat lid (102 &#176;C), 75 &#176;C for 15 min, 60 &#176;C for 10 min, 50 &#176;C for 10 min, 40 &#176;C for 10 min, 25 &#176;C for 30 min. 
	NOTE: The adapters are partially double-stranded DNA molecules (concentration = 50 pmol/&#181;L).</li>
	<li>Set up the following reaction in a 1.5 mL centrifuge tube: 10 &#181;L of pA-Tn5 transposase (500 ng/&#181;L or 7.5 pmol/&#181;L), 0.75 &#181;L of adaptor AB (50 pmol/&#181;L), 0.75 &#181;L of adaptor AC (50 pmol/&#181;L), 7 &#181;L of coupling buffer. Pipette 20x gently to mix well and incubate at 30 &#176;C in a water bath for 1 h. The final concentration of transposase = 4 pmol/&#181;L. Store at -20 &#176;C until use. 
	&#8203;NOTE: Molar ratio of adaptor AB: adaptor AC: transposase = 0.5:0.5:1.</li>
	<li>Prepare the stock solutions according to the recipes provided in Table 2 and autoclave or filter the stock solutions to sterilize.</li>
	<li>Autoclave and sterilize scissors, filter paper, a stainless-steel sieve, a mortar, pestles, etc.</li>
</ol>
2. Nuclei isolation (Day 2)
<ol>
	<li>Pre-cool the centrifuge to 4 &#176;C. For each 1 g of the starting material (cotton leaves), prepare 25 mL of nuclear isolation buffer A (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA), 20 mL of nuclear isolation buffer B (10 mM Tris pH 8.0, 50 mM NaCl, 1 mM EDTA), 50 mL of nuclear wash buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%), 1 mL of antibody buffer (50 mM Tris pH 8.0, 1 mM EDTA, 150 mM NaCl, 0.5 mM spermidine, 1 mg/mL BSA, protease inhibitor cocktail 0.1%, 0.05% w/v digitonin). Let the tubes sit on ice until use.</li>
	<li>Grind fresh leaves (~1 g) in liquid nitrogen to a fine powder and transfer to a 50 mL tube containing 25 mL of chilled nuclear isolation buffer A.</li>
	<li>Mix the buffer solution gently and incubate the tube on ice for 5 min with gentle shaking to disperse the material evenly.</li>
	<li>Centrifuge for 5 min at 500 rcf at 4 &#176;C to form the cell pellet.</li>
	<li>Decant the supernatant and resuspend the pellet with 20 mL of chilled nuclear isolation buffer B supplemented with 0.5% Triton X-100. Invert the tube gently to resuspend the pellet completely. 
	NOTE: The 20 mL nuclear isolation buffer B is applicable for 1 g starting material, and this volume can be scaled accordingly.
	<ol>
		<li>Perform a pilot assay to test the effect of series concentration of Triton X-100 (e.g., 2 mL nuclear isolation buffer B, each with 0.1%, 0.25%, 0.5%, and 1% Triton X-100 for 100 mg of grinded tissues). The appropriate concentration of Triton X-100 is indicated by an accumulation of white/gray nuclei at the bottom and a dark green supernatant after lysis and centrifuging at low speed (&#60; 500 rcf for 4 min).</li>
	</ol>
	</li>
	<li>Filter the resulting cell lysate with a combination of two sieves: a 500-mesh sieve on the top to remove the larger tissue debris and a 1000-mesh sieve on the bottom to collect the nuclei. 
	NOTE: Choose an appropriate mesh size for the sieves depending on the nuclei size of the plants.</li>
	<li>Use sterile filter paper on the back side of the sieve to absorb small cell debris in the lysate.</li>
	<li>Transfer the remaining nuclei containing lysate on the top side of the 1000-mesh sieve to four fresh 1.5 mL centrifuge tubes.</li>
	<li>Centrifuge for 4 min at 300 rcf at 4 &#176;C to collect the nuclei.</li>
	<li>Remove as much of the supernatant as possible using a pipette tip and wash the pellet with nuclear wash buffer.</li>
	<li>Add 800 &#181;L of the nuclear wash buffer and invert the tubes gently to resuspend the pellet. Spin the tube again for 4 min at 300 rcf at 4 &#176;C.</li>
	<li>Perform a total of 3 washes.</li>
	<li>(Optional) Image the nuclei under a microscope for DAPI staining.
	<ol>
		<li>Transfer 100 &#181;L of nuclei suspension in the nuclear wash buffer from Step 2.11. to a new 1.5 mL tube. Add 900 &#181;L of nuclear wash buffer and 2 &#181;L of DAPI stock solution (1 mg/mL) to make a final working concentration of 2 &#181;g/mL for DAPI. Incubate the tube in the dark for 30 min at room temperature.</li>
		<li>After staining, centrifuge for 4 min at 300 rcf at 4 &#176;C to collect the nuclei.</li>
		<li>Remove as much of the supernatant as possible using a pipette tip. Wash 3 times with 800 &#181;L of the nuclear wash buffer.</li>
		<li>Remove as much of the supernatant as possible and resuspend the nuclei with 100 &#181;L of nuclear wash buffer.</li>
		<li>Image the nuclei under a fluorescence microscope using the DAPI channel.</li>
	</ol>
	</li>
	<li>Combine the nuclei from two tubes (~50 &#181;L of nuclei in volume in each 1.5 mL tube). Centrifuge for 4 min at 300 rcf at 4 &#176;C. Remove as much of the remaining buffer as possible using a pipette tip.</li>
</ol>
3. Antibody incubation
<ol>
	<li>Resuspend each 50 &#181;L of nuclei in a 1.5 mL tube with 1 mL of antibody buffer.</li>
	<li>Set up the following reactions in 1.5 mL tubes: two biological replicates of IgG control groups with 150 &#181;L of nuclei (~ 1 &#181;g of chromatin) and 2 &#181;L of IgG (1 mg/mL) in each tube, two biological replicates of H3K4me3 assay groups with 150 &#181;L of nuclei and 2 &#181;L of anti-H3K4me3 antibody (1 mg/mL) in each tube.</li>
	<li>Mix the solution gently and leave the tubes on a horizontal shaker for hybridization overnight at 4 &#176;C and 12 rpm.</li>
	<li>Pre-cool the centrifuge to 4 &#176;C. Prepare fresh 50 mL of immunoprecipitation (IP) wash buffer (10 mM Tris pH 8.0, 150 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%, ,0.05% w/v digitonin) in a 50 mL centrifuge tube. Let the tube sit on ice.</li>
	<li>Centrifuge the tubes for 4 min at 300 rcf at 4 &#176;C. Remove the antibody buffer from each tube.</li>
	<li>Add 800 &#181;L of IP wash buffer to each tube and mix gently. Leave the tubes on a horizontal shaker for 5 min at room temperature and 12 rpm.</li>
	<li>After washing, centrifuge the tubes for 4 min at 300 rcf at 4 &#176;C. Remove the supernatant using a pipette tip. 
	NOTE: To avoid any disturbance of the nuclei pellet, leave ~50-80 &#181;L of the buffer with the nuclei.</li>
	<li>Repeat Steps 3.6.-3.7. two more times.</li>
	<li>After the third wash, centrifuge for 2 min at 300 rcf at 4 &#176;C. Remove the remaining buffer as much as possible.</li>
</ol>
4. Transposase incubation
<ol>
	<li>Make fresh 1 mL of transposase incubation buffer (10 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%, 0.05% w/v digitonin) by mixing 1 mL of the IP wash buffer with 30 &#181;L of 5 M NaCl.</li>
	<li>To prepare the Tn5 transposase mix for incubation, add 1 &#181;L of the transposase to each 150 &#181;L of the transposase incubation buffer. 
	&#8203;NOTE: Prepare the transposase solution mix first according to the number of reactions, and then add an aliquot of 150 &#181;L to each tube.</li>
	<li>Resuspend the nuclei with 150 &#181;L of transposase solution.</li>
	<li>Leave the tubes on a horizontal shaker at 12 rpm for 2-3 h at room temperature and mix every 10-15 min.</li>
	<li>After transposase incubation, centrifuge the tubes for 4 min at 300 rcf at 4 &#176;C. Remove the transposase incubation buffer in each tube.</li>
	<li>Wash the tubes with IP wash buffer. To do so, add 800 &#181;L of IP wash buffer to each tube, mix gently, and leave the tubes on a horizontal shaker at 12 rpm for 5 min at room temperature.</li>
	<li>Centrifuge for 4 min at 300 rcf at 4 &#176;C. Remove the supernatant using a pipette tip.</li>
	<li>Repeat Steps 4.6.-4.7. two more times.</li>
	<li>After the third wash, centrifuge for 2 min at 300 rcf at 4 &#176;C. Remove the remaining buffer as much as possible.</li>
</ol>
5. Tagmentation
<ol>
	<li>Freshly make 2 mL of the tagmentation buffer (10 mM Tris pH 8.0, 300 mM NaCl, 0.5 mM spermidine, protease inhibitor cocktail 0.1%, 10 mM MgCl2, 0.05% w/v digitonin) by mixing 2 mL of IP wash buffer with 60 &#181;L of 5 M NaCl and 20 &#181;L of 1 M MgCl2.</li>
	<li>Resuspend the nuclei with 300 &#181;L of tagmentation buffer.</li>
	<li>Mix the solution and incubate in a 37 &#176;C water bath for 1 h for tagmentation.</li>
	<li>Stop the tagmentation with 10 &#181;L of 0.5 M EDTA (final concentration ~15 mM) and 30 &#181;L of 10% SDS (final concentration 1%).</li>
</ol>
6. DNA extraction and NGS library construction
<ol>
	<li>Add 300 &#181;L of CTAB DNA extraction buffer as described7. Incubate the tubes in a 65 &#176;C water bath for 30 min. Invert to mix every ~10 min.</li>
	<li>Add 600 &#181;L of phenol: chloroform: isoamyl alcohol (25:24:1) to each tube. Mix thoroughly.</li>
	<li>Pre-centrifuge the phase lock gel tubes for 2 min at 13,000 rcf at 4 &#176;C. Transfer the above solution to phase lock gel tubes.</li>
	<li>Centrifuge for 10 min at 13,000 rcf at 4 &#176;C.</li>
	<li>Transfer the supernatant (~600 &#181;L) to a new 1.5 mL tube.</li>
	<li>Add 600 &#181;L of chloroform to each tube. Mix thoroughly.</li>
	<li>Centrifuge for 10 min at 13,000 rcf at 4 &#176;C.</li>
	<li>Transfer the supernatant (~600 &#181;L) to a new 1.5 ml tube.</li>
	<li>Add 12 &#181;L of 100% ethanol and 2 &#181;L of GlycoBlue Coprecipitant. Mix thoroughly and store at -20 &#176;C for 1 h.</li>
	<li>Centrifuge for 10 min at 13,000 rcf at 4 &#176;C.</li>
	<li>Decant the supernatant. Wash the DNA pellet using 75% (v/v) ethanol and centrifuge for 5 min at 13,000 rcf at 4 &#176;C.</li>
	<li>Decant the supernatant. Air-dry the DNA pellet and resuspend the DNA in 25 &#181;L of ddH2O.</li>
	<li>Set up the PCR reaction as follows. For a total of 50 &#181;L of reaction, add 24 &#181;L of DNA, 11 &#181;L of ddH2O, 10 &#181;L of 5x TAE buffer, 2 &#181;L of P5 primer (10 &#181;M), 2 &#181;L of P7 primer (10 &#181;M), and 1 &#181;L of TruePrep Amplify Enzyme. PCR program: 72 &#176;C for 3 min, 98 &#176;C for 30 s; then 14 cycles of 98 &#176;C for 30 s, 60 &#176;C for 30 s, and 72 &#176;C for 30 s, followed by 72 &#176;C for 5 min. 
	NOTE: The first 72 &#176;C for 3 min for extension cannot be skipped. Overamplification of the library will lead to a high level of PCR duplicates in the NGS. Start from 12-14 PCR cycles in PCR reaction for H3K4me3 when using ~1 &#181;g of chromatin in each reaction.</li>
	<li>Load 2 &#181;L of the PCR products on 1.5% agarose gel for electrophoresis.</li>
	<li>Purify the PCR products using commercial DNA purification magnetic beads.</li>
	<li>Quantify the library with a fluorometer and agarose gel electrophoresis. 
	NOTE: Effective concentration of the library should be &#62;2 nM by qPCR to ensure good quality.</li>
	<li>Perform next-generation sequencing (NGS) and data analysis.</li>
</ol>

<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>

All authors have no conflicts of interest with any company trading one of the products mentioned above.

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><tbody><tr><td>&lt;strong&gt;Antibody&lt;/strong&gt;</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>&lt;strong&gt;Chemicals&lt;/strong&gt;</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&amp;deg;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&amp;deg;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 (MgCl&lt;sub&gt;2&lt;/sub&gt;)</td><td></td><td></td><td>Make 1 M MgCl&lt;sub&gt;2&lt;/sub&gt; 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&amp;deg;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>&lt;strong&gt;Enzyme&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Hyperactive pG-Tn5/pA-Tn5 transposase for CUT&amp;amp;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>&lt;strong&gt;Equipment&lt;/strong&gt;</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&amp;nbsp; 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 ...

profiling-h3k4me3-modification-plants-using-cleavage-under-targets

Research

JoVE Journal

Biology

3.9K Views.  Zhejiang University. Het algemene doel van het experiment was om eiwit-geaffilieerde DNA-fragmenten in het genoom te identificeren, die de status van het chromatine weerspiegelden. Om dit doel te bereiken werden drie speciale operaties uitgevoerd. Allereerst werden de intacte kernen van plantencellen geïsoleerd.Ten tweede werden modificaties aan het chromatine herkend door een specifiek antilichaam in situ. Ten derde, het antilichaam dat een eiwit A-transposase fusie-eiwit, Tn5 transposase, bindt, splitst...

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

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription factors (TFs), chromatin regulators, and histone modifications, as well as detecting entire genomes for uncovering TF binding patterns and histone posttranslational modifications. Chromatin-modifying activities, such as histone methylation, are often recruited to specific gene regulatory sequences, causing localized changes in chromatin structures and resulting in specific transcriptional effects. The rice blast is a devastating fungal disease on rice throughout the world and is a model system for studying fungus-plant interaction. However, the molecular mechanisms in how the histone modifications regulate their virulence genes in Magnaporthe oryzae remain elusive. More researchers need to use ChIP-seq to study how histone epigenetic modification regulates their target genes. ChIP-seq is also widely used to study the interaction between protein and DNA in animals and plants, but it is less used in the field of plant pathology and has not been well developed. In this paper, we describe the experimental process and operation method of ChIP-seq to identify the genome-wide distribution of histone methylation (such as H3K4me3) that binds to the functional target genes in M. oryzae. Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae

Here, we present a protocol to analyze the genome-wide distribution of histone modifications, which can identify new target genes in the pathogenesis of M. oryzae and other filamentous fungi.

Chromatin immunoprecipitation sequencing (ChIP-seq) is a powerful and widely used molecular technique for mapping whole genome locations of transcription ...

Genetics

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Biochemistry

Histone Proteomic Analysis and Post-Translational Modification Characterization

Histone Modification Screening using Liquid Chromatography, Trapped Ion Mobility Spectrometry, and Time-Of-Flight Mass Spectrometry

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as posttranslational modifications (PTMs). Identifying the position and nature of each PTM or pattern of PTMs in reference to external or genetic factors allows this information to be statistically correlated with biological responses such as DNA transcription, replication, or repair. In the present work, a high-throughput analytical protocol for the detection of histone PTMs from biological samples is described. The use of complementary liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) enables the separation and PTM assignment of the most biologically relevant modifications in a single analysis. The described approach takes advantage of recent developments in dependent data acquisition (DDA) using parallel accumulation in the mobility trap, followed by sequential fragmentation and collision-induced dissociation. Histone PTMs are confidently assigned based on their retention time, mobility, and fragmentation pattern.

Author Spotlight: Enhanced Histone PTM Isomer Identification Through LC-TIMS-ToF MS/MS and PASEF

An analytical workflow based on liquid chromatography, trapped ion mobility spectrometry, and time-of-flight mass spectrometry (LC-TIMS-ToF MS/MS) for high confidence and highly reproducible "bottom-up" analysis of histone modifications and identification based on principal parameters (retention time [RT], collision cross section [CCS], and accurate mass-to-charge [m/z] ratio).

Histone proteins are highly abundant and conserved among eukaryotes and play a large role in gene regulation as a result of structures known as ...

Chemistry

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Profiling Genomic Histone Marks in Mouse Myoblasts Using CUT&#38;Tag

Using Cleavage Under Targets and Tagmentation (CUT&#38;Tag) Assay in Mouse Myoblast Research

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile the genomic locations of chromatin binding factors, histone marks, and histone variants. CUT&amp;Tag protocols function very well with mouse myoblasts and freshly isolated muscle stem cells (MuSCs). They can easily be applied to many other cell types as long as the cells can be immobilized by Concanavalin-A beads. Compared to CUT&amp;Tag, chromatin immunoprecipitation (ChIP) assays are time-consuming experiments. ChIP assays require the pre-treatment of chromatin before the chromatic material can be used for immunoprecipitation. In cross-linking ChIP (X-ChIP), pre-treatment of chromatin involves cross-linking and sonication to fragment the chromatin. In the case of native ChIP (N-ChIP), the fragmented chromatins are normally achieved by Micrococcal nuclease (MNase) digestion. Both sonication and MNase digestion introduce some bias to the ChIP experiments. CUT&amp;Tag assays can be finished within fewer steps and require much fewer cells compared to ChIPs but provide more unbiased information on transcription factors or histone marks at various genomic locations. CUT&amp;Tag can function with as few as 5,000 cells. Due to its higher sensitivity and lower background signal than ChIPs, researchers can expect to obtain reliable peak data from merely several millions of reads after sequencing.

Author Spotlight: Navigating Challenges and Innovations in Muscle Stem Cell Studies

Researchers new to the epigenetic field will find CUT&amp;Tag a significantly easier alternative to ChIP assays. CUT&amp;Tag has tremendously benefited the epigenetic studies on rare and primary cell populations, generating high-quality data from very few cells. This protocol describes performing H3K4me1 CUT&amp;Tag assays on mouse myoblasts isolated from mouse hindlimb muscles.

This protocol paper aims to provide the new researchers with the full details of using Cleavage Under Targets and Tagmentation (CUT&amp;Tag) to profile ...

Developmental Biology

Detection of Histone Modifications in Plant Leaves

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent modifications on the amino-terminal tails of histones H3 and H4 play essential roles1-2. H3 and H4 histone modifications include methylation of lysine and arginine, acetylation of lysine, and phosphorylation of serine residues1-2. These modifications are associated either with gene activation, repression, or a primed state of gene that supports more rapid and robust activation of expression after perception of appropriate signals (microbe-associated molecular patterns, light, hormones, etc.)3-7. 
Here, we present a method for the reliable and sensitive detection of specific chromatin modifications on selected plant genes. The technique is based on the crosslinking of (modified) histones and DNA with formaldehyde8,9, extraction and sonication of chromatin, chromatin immunoprecipitation (ChIP) with modification-specific antibodies9,10, de-crosslinking of histone-DNA complexes, and gene-specific real-time quantitative PCR. The approach has proven useful for detecting specific histone modifications associated with C4 photosynthesis in maize5,11 and systemic immunity in Arabidopsis3.

A reliable and useful approach to detect histone modifications on specific plant genes is described. The approach combines chromatin immunoprecipitation (ChIP) and real-time quantitative PCR. It allows detection of histone modifications on specific genes with a role in diverse physiological processes.

Chromatin structure is important for the regulation of gene expression in eukaryotes. In this process, chromatin remodeling, DNA methylation, and covalent ...

Isolation and Cultivation of Neural Progenitors Followed by Chromatin-Immunoprecipitation of Histone 3 Lysine 79 Dimethylation Mark

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. Additionally, epigenetic chromatin modifications, like histone methylation, have an important role for establishing and maintaining specific cell fates within this process. The vast majority of histone methylation occurs on the flexible histone tail, which is accessible to histone modifiers, erasers, and histone reader proteins. In contrast, H3K79 methylation is located in the globular domain of histone 3 and is implicated in different developmental functions. H3K79 methylation is evolutionarily conserved and can be found in a wide range of species from Homo sapiens to Saccharomyces cerevisiae. The modification occurs in different cell populations within organisms, including neural progenitors. The location of H3K79 methylation in the globular domain of histone 3 makes it difficult to assess. Here, we present methods to isolate and culture cortical progenitor cells (CPCs) from embryonic cortical brain tissue (E11.5-E14.5) or cerebellar granular neuron progenitors (CGNPs) from postnatal tissue (P5-P7), and to efficiently immunoprecipitate H3K79me2 for quantitative PCR (qPCR) and genome-wide sequencing.

We present an effective and reproducible method to isolate and culture neural progenitor cells from embryonic and postnatal brain tissue for chromatin immunoprecipitation (ChIP) of histone 3 lysine 79 dimethylation (H3K79me2) - a histone mark located within the globular domain of histone 3.

Brain development is a complex process, which is controlled in a temporo-spatial manner by gradients of morphogens and different transcriptional programs. ...

Neuroscience

The ChroP Approach Combines ChIP and Mass Spectrometry to Dissect Locus-specific Proteomic Landscapes of Chromatin

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and function at specific regions is regulated by the local enrichment of histone post-translational modifications (hPTMs) and variants, chromatin-binding proteins, including transcription factors, and DNA methylation. The proteomic characterization of chromatin composition at distinct functional regions has been so far hampered by the lack of efficient protocols to enrich such domains at the appropriate purity and amount for the subsequent in-depth analysis by Mass Spectrometry (MS). We describe here a newly designed chromatin proteomics strategy, named ChroP (Chromatin Proteomics), whereby a preparative chromatin immunoprecipitation is used to isolate distinct chromatin regions whose features, in terms of hPTMs, variants and co-associated non-histonic proteins, are analyzed by MS. We illustrate here the setting up of ChroP for the enrichment and analysis of transcriptionally silent heterochromatic regions, marked by the presence of tri-methylation of lysine 9 on histone H3. The results achieved demonstrate the potential of ChroP in thoroughly characterizing the heterochromatin proteome and prove it as a powerful analytical strategy for understanding how the distinct protein determinants of chromatin interact and synergize to establish locus-specific structural and functional configurations.

By combining native and crosslinking chromatin immunoprecipitation with high-resolution Mass Spectrometry, ChroP approach enables to dissect the composite proteomic architecture of histone modifications, variants and non-histonic proteins synergizing at functionally distinct chromatin domains.

Chromatin is a highly dynamic nucleoprotein complex made of DNA and proteins that controls various DNA-dependent processes. Chromatin structure and ...

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the adult plant. The female SMC (megaspore mother cell, MMC) differentiates in the ovule primordium and undergoes meiosis. The selected haploid megaspore then undergoes mitosis to form the multicellular female gametophyte, which will give rise to the gametes, the egg cell and central cell, together with accessory cells. The limited accessibility of the MMC, meiocyte and female gametophyte inside the ovule is technically challenging for cytological and cytogenetic analyses at single cell level. Particularly, direct or indirect immunodetection of cellular or nuclear epitopes is impaired by poor penetration of the reagents inside the plant cell and single-cell imaging is demised by the lack of optical clarity in whole-mount tissues.
Thus, we developed an efficient method to analyze the nuclear organization and chromatin modification at high resolution of single cell in whole-mount embedded Arabidopsis ovules. It is based on dissection and embedding of fixed ovules in a thin layer of acrylamide gel on a microscopic slide. The embedded ovules are subjected to chemical and enzymatic treatments aiming at improving tissue clarity and permeability to the immunostaining reagents. Those treatments preserve cellular and chromatin organization, DNA and protein epitopes. The samples can be used for different downstream cytological analyses, including chromatin immunostaining, fluorescence in situ hybridization (FISH), and DNA staining for heterochromatin analysis. Confocal laser scanning microscopy (CLSM) imaging, with high resolution, followed by 3D reconstruction allows for quantitative measurements at single-cell resolution.

An Efficient Method for Quantitative, Single-cell Analysis of Chromatin Modification and Nuclear Architecture in Whole-mount Ovules in Arabidopsis

We provide here an efficient and reliable protocol for immunostaining, Fluorescence in&#160;situ Hybridization, DNA staining followed by quantitative, high-resolution imaging in whole-mount Arabidopsis thaliana ovules. This method was successfully used to analyze chromatin modifications and nuclear architecture.

In flowering plants, the somatic-to-reproductive cell fate transition is marked by the specification of spore mother cells (SMCs) in floral organs of the ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

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

Samenvatting

Meer video's verkennen

Hoofdstukken in deze video