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 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...

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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 ...

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3.9K Views.  Zhejiang University. Cleavage under targets and tagmentation (CUT&Tag) is an efficient chromatin epigenomic profiling strategy. This protocol presents a refined CUT&Tag strategy for the profiling of histone modifications in plants. 

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

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage (CoTrAC)

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method makes it possible to count the numbers of protein molecules produced in one cell during sequential, five-minute time windows. It requires a fluorescence microscope with laser excitation power density of ~0.5 to 1 kW/cm2, which is sufficiently sensitive to detect single fluorescent protein molecules in live cells. The fluorescent reporter used in this method consists of three parts: a membrane targeting sequence, a fast-maturing, yellow fluorescent protein and a protease recognition sequence. The reporter is translationally fused to the N-terminus of a protein of interest. Cells are grown on a temperature-controlled microscope stage. Every five minutes, fluorescent molecules within cells are imaged (and later counted by analyzing fluorescence images) and subsequently photobleached so that only newly translated proteins are counted in the next measurement.
Fluorescence images resulting from this method can be analyzed by detecting fluorescent spots in each image, assigning them to individual cells and then assigning cells to cell lineages. The number of proteins produced within a time window in a given cell is calculated by dividing the integrated fluorescence intensity of spots by the average intensity of single fluorescent molecules. We used this method to measure expression levels in the range of 0-45 molecules in single 5 min time windows. This method enabled us to measure noise in the expression of the &lambda; repressor CI, and has many other potential applications in systems biology.

Single-molecule Imaging of Gene Regulation In vivo Using Cotranslational Activation by Cleavage CoTrAC

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC), to image the production of protein molecules in live cells with single-molecule precision without perturbing the protein's functionality. This method has been used to follow the stochastic expression dynamics of a transcription factor, the &lambda; repressor CI 1.

We describe a fluorescence microscopy method, Co-Translational Activation by Cleavage (CoTrAC) to image the production of protein molecules in live cells ...

Analysis of RNA Processing Reactions Using Cell Free Systems: 3' End Cleavage of Pre-mRNA Substrates in vitro

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes precursor mRNA beyond the end of mature RNAs, and an active process of endonuclease activity is required at a specific site. Cleavage of the precursor RNA normally occurs 10-30 nt downstream from the consensus polyA site (AAUAAA) after the CA dinucleotides. Proteins from the cleavage complex, a multifactorial protein complex of approximately 800 kDa, accomplish this specific nuclease activity. Specific RNA sequences upstream and downstream of the polyA site control the recruitment of the cleavage complex. Immediately after cleavage, pre-mRNAs are polyadenylated by the polyA polymerase (PAP) to produce mature stable RNA messages.
Processing of the 3&#8217; end of an RNA transcript may be studied using cellular nuclear extracts with specific radiolabeled RNA substrates. In sum, a long 32P-labeled uncleaved precursor RNA is incubated with nuclear extracts in vitro, and cleavage is assessed by gel electrophoresis and autoradiography. When proper cleavage occurs, a shorter 5&#8217; cleaved product is detected and quantified. Here, we describe the cleavage assay in detail using, as an example, the 3&#8217; end processing of HIV-1 mRNAs.

Analysis of RNA Processing Reactions Using Cell Free Systems: 3&#039; End Cleavage of Pre-mRNA Substrates in vitro

RNA polymerase II synthesizes a precursor RNA that extends beyond the 3' end of the mature mRNA. The end of the mature RNA is generated cotranscriptionally, at a site dictated by RNA sequences, via the endonuclease activity of the cleavage complex. Here, we detail the method to study cleavage reactions in vitro.

The 3&#8217; end of mammalian mRNAs is not formed by abrupt termination of transcription by RNA polymerase II (RNPII). Instead, RNPII synthesizes ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ binding proteins that initiate Ca2+ signaling cascades. However, we still know little about how stimulus specific Ca2+ signals are generated. The specificity of a Ca2+ signal may be attributed to the sophisticated regulation of the activities of Ca2+ channels and/or transporters in response to a given stimulus. To identify these cellular components and understand their functions, it is crucial to use systems that allow a sensitive and robust recording of Ca2+ signals at both the tissue and cellular levels. Genetically encoded Ca2+ indicators that are targeted to different cellular compartments have provided a platform for live cell confocal imaging of cellular Ca2+ signals. Here we describe instructions for the use of two Ca2+ detection systems: aequorin based FAS (film adhesive seedlings) luminescence Ca2+ imaging and case12 based live cell confocal fluorescence Ca2+ imaging. Luminescence imaging using the FAS system provides a simple, robust and sensitive detection of spatial and temporal Ca2+ signals at the tissue level, while live cell confocal imaging using Case12 provides simultaneous detection of cytosolic and nuclear Ca2+ signals at a high resolution.

Measuring Spatial and Temporal Ca2+ Signals in Arabidopsis Plants

Ca2+ signaling regulates diverse biological processes in plants. Here we present approaches for monitoring abiotic stress induced spatial and temporal Ca2+ signals in Arabidopsis cells and tissues using the genetically encoded Ca2+ indicators Aequorin or Case12.

Developmental and environmental cues induce Ca2+ fluctuations in plant cells. Stimulus-specific spatial-temporal Ca2+ patterns are sensed by cellular Ca2+ ...

Detection of miRNA Targets in High-throughput Using the 3'LIFE Assay

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an array of hundreds of queried 3&#8217;UTRs. Target identification is based on the dual-luciferase assay, which detects binding at the mRNA level by measuring translational output, giving a functional readout of miRNA targeting. 3&#8217;LIFE uses non-proprietary buffers and reagents, and publically available reporter libraries, making genome-wide screens feasible and cost-effective. 3&#8217;LIFE can be performed either in a standard lab setting or scaled up using liquid handling robots and other high-throughput instrumentation. We illustrate the approach using a dataset of human 3&#8217;UTRs cloned in 96-well plates, and two test miRNAs, let-7c and miR-10b. We demonstrate how to perform DNA preparation, transfection, cell culture and luciferase assays in 96-well format, and provide tools for data analysis. In conclusion 3&#39;LIFE is highly reproducible, rapid, systematic, and identifies high confidence targets.

Detection of miRNA Targets in High-throughput Using the 3&#039;LIFE Assay

Luminescent identification of functional elements in 3&#8217; untranslated regions (3&#8217;UTRs) (3&#8217;LIFE) is a technique to identify functional regulation in 3&#8217;UTRs by miRNAs or other regulatory factors. This protocol utilizes high-throughput methodology such as 96-well transfection and luciferase assays to screen hundreds of putative interactions for functional repression.

Luminescent Identification of Functional Elements in 3&#8217;UTRs (3&#8217;LIFE) allows the rapid identification of targets of specific miRNAs within an ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...

Genetic Modification of Cyanobacteria by Conjugation Using the CyanoGate Modular Cloning Toolkit

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful industrial commodities. Recent advances in synthetic biology have led to development of several cloning toolkits such as CyanoGate, a standardized modular cloning system for building plasmid vectors for subsequent transformation or conjugal transfer into cyanobacteria. Here we outline a detailed method for assembling a self-replicating vector (e.g., carrying a fluorescent marker expression cassette) and conjugal transfer of the vector into the cyanobacterial strains Synechocystis sp. PCC 6803 or Synechococcus elongatus UTEX 2973. In addition, we outline how to characterize the performance of a genetic part (e.g., a promoter) using a plate reader or flow cytometry.

Here, we present a protocol describing how to i) assemble a self-replicating vector using the CyanoGate modular cloning toolkit, ii) introduce the vector into a cyanobacterial host by conjugation, and iii) characterize transgenic cyanobacteria strains using a plate reader or flow cytometry.

Cyanobacteria are a diverse group of prokaryotic photosynthetic organisms that can be genetically modified for the renewable production of useful ...

Single-Cell Factor Localization on Chromatin using Ultra-Low Input Cleavage Under Targets and Release using Nuclease

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin Immunoprecipitation (ChIP) has been the gold standard for determining protein localization for over 30 years and is defined by the use of an antibody to pull out the protein of interest from sonicated or enzymatically digested chromatin. More recently, antibody tethering techniques have become popular for assessing protein localization on chromatin due to their increased sensitivity. Cleavage Under Targets &amp; Release Under Nuclease (CUT&amp;RUN) is the genome-wide derivative of Chromatin Immunocleavage (ChIC) and utilizes recombinant Protein A tethered to micrococcal nuclease (pA-MNase) to identify the IgG constant region of the antibody targeting a protein of interest, therefore enabling site-specific cleavage of the DNA flanking the protein of interest. CUT&amp;RUN can be used to profile histone modifications, transcription factors, and other chromatin-binding proteins such as nucleosome remodeling factors. Importantly, CUT&amp;RUN can be used to assess the localization of either euchromatic- or heterochromatic-associated proteins and histone modifications. For these reasons, CUT&amp;RUN is a powerful method for determining the binding profiles of a wide range of proteins. Recently, CUT&amp;RUN has been optimized for transcription factor profiling in low populations of cells and single cells and the optimized protocol has been termed ultra-low input CUT&amp;RUN (uliCUT&amp;RUN). Here, a detailed protocol is presented for single-cell factor profiling using uliCUT&amp;RUN in a manual 96-well format.

CUT&#38;RUN and its variants can be used to determine protein occupancy on chromatin. This protocol describes how to determine protein localization on chromatin using single-cell uliCUT&#38;RUN.

Determining the binding locations of a protein on chromatin is essential for understanding its function and potential regulatory targets. Chromatin ...

Microscopy Techniques for Interpreting Fungal Colonization in Mycoheterotrophic Plants Tissues and Symbiotic Germination of Seeds

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching mycoheterotrophic plants (i.e., plants that obtain carbon from fungi), remarkable aspects of their structural adaptations, the patterns of tissue colonization by fungi, and the morphoanatomy of subterranean organs can enlighten their developmental strategies and their relationships with hyphae, the source of nutrients. Another important role of symbiotic fungi is related to the germination of orchid seeds; all Orchidaceae species are mycoheterotrophic during germination and seedling stage (initial mycoheterotrophy), even the ones that photosynthesize in adult stages. Due to the lack of nutritional reserves in orchid seeds, fungal symbionts are essential to provide substrates and enable germination. Analyzing germination stages by structural perspectives can also answer important questions regarding the fungi interaction with the seeds. Different imaging techniques can be applied to unveil fungi endophytes in plant tissues, as are proposed in this article. Freehand and thin sections of plant organs can be stained and then observed using light microscopy. A fluorochrome conjugated to wheat germ agglutinin can be applied to the fungi and co-incubated with Calcofluor White to highlight plant cell walls in confocal microscopy. In addition, the methodologies of scanning and transmission electron microscopy are detailed for mycoheterotrophic orchids, and the possibilities of applying such protocols in related plants is explored. Symbiotic germination of orchid seeds (i.e., in the presence of mycorrhizal fungi) is described in the protocol in detail, along with possibilities of preparing the structures obtained from different stages of germination for analyses with light, confocal, and electron microscopy.

This protocol aims to provide detailed procedures for collecting, fixing, and maintaining mycoheterotrophic plant samples, applying different microscopy techniques such as scanning and transmission electron microscopy, light, confocal, and fluorescence microscopy to study fungal colonization in plants tissues and seeds germinated with mycorrhizal fungi.

Structural botany is an indispensable perspective to fully understand the ecology, physiology, development, and evolution of plants. When researching ...