This work was supported by National Natural Science Foundation of China (Grant no. 31871638), the Special Scientific Research Project of Beijing Agriculture University (YQ201603), the Scientific Project of Beijing Educational Committee (KM201610020005), the High-level scientific research cultivation project of BUA (GJB2021005).

1. Preparation of protoplasts from M. oryzae
<ol>
	<li>Prepare the oatmeal-tomato agar (OTA).
	<ol>
		<li>Weigh 30-50 g of oatmeal and boil it in 800 mL of water (ddH2O) for 20 min. Filter through two layers of gauze and take the filtrate.</li>
		<li>Pick ripe tomatoes and peel them. Squeeze the juice, and filter through two layers of gauze to collect 150 mL of the filtered juice.</li>
		<li>Mix all the tomato juice and the prepared oat filtrate thoroughly and add ddH2O up to 1000 mL.</li>
		<li>Add 250 mL of the OTA and 2.5 g of agar powder to a 500 mL conical flask, and autoclave for 20 min. Store at 25 &#176;C.</li>
	</ol>
	</li>
	<li>Pour 25 mL of the autoclaved OTA into a glass 5 cm x 5 cm Petri dish. Prepare 10 of these Petri dishes total. After the OTA has solidified on the Petri dishes, store the dishes upside down at 25 &#176;C.</li>
	<li>Use a sterilized toothpick to dig out a small piece of mycelium from M. oryzae (the wild-type strain P131, knockout strains &#916;mobre2, &#916;mospp1, and &#916;moswd2) and place them on the prepared OTA dishes. Culture them for 4-6 days at 28 &#176;C under light conditions. 
	NOTE: Turn the Petri dish upside down to prevent pollution.</li>
	<li>Add 1000 &#181;L of liquid Complete Medium (CM) (0.6% yeast extract, 0.3% enzymatic casein hydrolysate, 0.3% acidic casein hydrolysate, 1% glucose) to the OTA dishes using a 1000 &#181;L pipette. 
	NOTE: The hyphae grew on the OTA dishes for 4-6 days.</li>
	<li>Scrape the mycelia of the wild-type strain and knockout strain with an inoculation loop.</li>
	<li>Collect the mycelia debris and transfer them to 250 mL of liquid Complete Medium (CM).</li>
	<li>Grow the fungal debris in a triangular flask at 28 &#176;C for 36 h with shaking at 150 rpm.</li>
	<li>Use a funnel to filter and collect the fungal hyphae.</li>
	<li>Wash the fungal hyphae with 500 mL of 0.7 M NaCl solution.</li>
	<li>Collect the fungal mycelium and weigh it. 
	NOTE: The mycelium does not need to be dried before weighing.</li>
	<li>Add ~1 mL of lysis enzyme permeation solution per 1 g of the fungal mycelium.
	<ol>
		<li>Prepare the 20 mg/mL lysis enzyme permeation solution by dissolving the lysis enzyme from Trichoderma harzianum in 0.7 M NaCl.</li>
	</ol>
	</li>
	<li>Place the hyphae for lysing at 28 &#176;C for 3-4 h with shaking at 150 rpm.</li>
	<li>Wash the lysed hyphae with 50 mL of 0.7 M NaCl solution.</li>
	<li>Collect the protoplasts and centrifuge for 15 min at 2,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant carefully. Resuspend the protoplasts in 20 mL of 0.7 M NaCI buffer at 4 &#176;C.</li>
</ol>
2. In vivo crosslinking and sonication
<ol>
	<li>Add 55 &#181;L of 37% formaldehyde (add formaldehyde drop by drop until the final concentration is 1%) to 2 mL of NaCI buffer containing protoplast for crosslinking.</li>
	<li>Incubate the protoplasts at25 &#176;Cfor 10 min.</li>
	<li>Add 20 &#181;L of 10x glycine to each tube to quench the unreacted formaldehyde.</li>
	<li>Swirl to mix and incubate at 25 &#176;C for 5 min.</li>
	<li>Centrifuge for 15 min at 2,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant carefully. Resuspend the pellet in 1 mL of 0.7 M NaCI solution.</li>
	<li>Centrifuge for 10 min at 2,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, discard the supernatant carefully. Resuspend the pellet in 750 &#181;L of RIPA buffer(50 mM Tris-HCl pH 8, 150 mM NaCl, 2 mM EDTA pH 8, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS, and protease inhibitors).</li>
	<li>Add 37.5 &#181;L of 20x protease inhibitor.</li>
	<li>Transfer the protoplasts from the previous step to a 1.5 mL centrifuge tube.</li>
	<li>Perform sonication (25% W, output 3 s, stop 5 s, 4 &#176;C) immediately with a sonicator for about 10 min. The purpose of this step is to sonicate the cross-linked lysate for a period of time to determine the best conditions. 
	NOTE: The lysate can be frozen at -80 &#176;C at this step.</li>
	<li>If optimal conditions for sonication have already been determined, proceed to the next step.</li>
	<li>If desired, remove 5 &#181;L of protoplasts for agarose gel analysis (unsheared DNA).</li>
	<li>Shear the chromatin by sonication with an ultrasonic homogenizer for 8 min (25% W, output 3 s, stop 5 s, 4 &#176;C).</li>
	<li>After the sample is ultrasonically broken, take out a part of the sample as &#34;input&#34;.. The input does not perform the ChIP experiment and contains all DNA and protein released after the sample is sonicated.</li>
	<li>After sonication, run a 1% agarose gel electrophoresis to analyze the length of the DNA fragments. 
	NOTE: The agarose gel electrophoresis results show that the length of the DNA fragment is 200-500 bp (Figure 4).</li>
	<li>Place the sonicated tube on ice to prevent protein degradation.</li>
	<li>Centrifuge for 10 min at 10,000 x g and 4 &#176;C.</li>
	<li>After centrifugation, transfer the centrifuged supernatant to a new 1.5 mL centrifuge tube and store it at -80 &#176;C for later use. The chromatin solution obtained in this step can be used for subsequent IP.</li>
	<li>Before performing the IP experiment, dilute each chromatin sample to a ratio of 1:10 with 1x RIPA buffer (e.g., add 10 &#181;L of chromatin sample to 1 &#181;L of 1&#215;RIPA buffer).</li>
</ol>
3. IP of crosslinked protein/DNA
<ol>
	<li>Pipette 50 &#181;L of superparamagnetic protein beads into a 2 mL centrifuge tube. Place the tubes on a magnetic stand. Let the magnetic beads precipitate. Remove the supernatant.</li>
	<li>Add 1 mL of 1x RIPA buffer (pre-cooled on ice) to the tube and wash superparamagnetic protein beads thrice. After the wash, place the tubes on a magnetic stand and remove the supernatant. Add 100 &#181;L of 1x RIPA buffer to each tube.</li>
	<li>Add 300 &#181;L of chormatin sample (2 x 107 cells were used), 100 &#181;L of superparamagnetic protein beads and 4 &#181;L of H3K4me3 antibody to the tube.</li>
	<li>Use samples with Mouse IgG as the negative control. 
	NOTE: Mouse IgG used in this protocol contains 0.01 M phosphate buffer and 0.15 M NaCl, and will remain frozen below 20 &#176;C (see Table of Materials).</li>
	<li>After mixing well, place the samples on a rotary shaker and incubate overnight at 4 &#176;C, 30 x g. 
	&#8203;NOTE: It may be possible to reduce the incubation time of the immunoprecipitation (IP). The incubation time depends on different factors (e.g., the antibody, gene target, cell type, etc.) and will need to be empirically tested.</li>
</ol>
4. Collecting and rinsing the IP products
<ol>
	<li>Pellet the superparamagnetic protein beads by placing them on a magnetic stand. Aspirate and discard the supernatant.</li>
	<li>Wash the superparamagnetic protein bead-antibody/chromatin complex by resuspending the beads in 1 mL of 1x RIPA buffer.</li>
	<li>Rinse the tube on a rotary shaker for 5 min and remove the supernatant at 30 x g.</li>
	<li>Add 1 mL of low salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, and 150 mM NaCl).</li>
	<li>Rinse the tube on a rotary shaker for 5 min and remove the supernatant.</li>
	<li>Add 1 mL of high salt immune complex wash buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl pH 8.1, and 500 mM NaCl) to the centrifuge tube.</li>
	<li>Place the tube on a rotary shaker for 5 min and remove the supernatant.</li>
	<li>Rinse with 1 mL of LiCl (0.25 M LiCl, 1% NP-40, 1% deoxycholic acid, 1 mM EDTA, and 10 mM Tris-HCl pH 8.1) in the centrifuge tube.</li>
	<li>Place the tube on a rotary shaker for 5 min and remove the supernatant.</li>
	<li>Rinse the test tube again with 1 mL of 0.25 M LiCI buffer, remove the supernatant with a pipette, and then discard the supernatant.</li>
	<li>Add 1 mL of TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA).</li>
	<li>Place the tube on a rotary shaker for 5 min at 30 x g.</li>
	<li>Wash the tube again with 1 mL TE buffer and remove the supernatant with a pipette.</li>
	<li>Collect the beads.</li>
</ol>
5. Elution of protein/DNA complexes
<ol>
	<li>Prepare the elution buffer (10 &#181;L of 20% SDS, 20 &#181;L of 1 M NaHCO3, 170 &#181;L of sterile distilled H2O) for all the IP and input tubes.</li>
	<li>Add 100 &#181;L of elution buffer to each centrifuge tube.</li>
	<li>Elution at 65 &#176;C for 15 min.</li>
	<li>Centrifuge for 1 min at 10,000 x g and 4 &#176;C and collect the supernatant into new centrifuge tubes.</li>
	<li>Repeat steps 5.2 - 5.4 and combine the eluates. Add 190 &#181;L of elution buffer to the 10 &#181;L of the input DNA. (total volume = 200 &#181;L).</li>
</ol>
6. Reverse crosslinking of protein/DNA complexes
<ol>
	<li>Add 8 &#181;L of 5 M NaCl to all the tubes and incubate at 65 &#176;C for 4-5 h or overnight to reverse the DNA-protein crosslinks. 
	NOTE: After this step, store the samples at -20 &#176;C, and continue the protocol the following day, if necessary.</li>
	<li>Add 1 &#181;L of RNase A and incubate for 30 min at 37 &#176;C.</li>
	<li>Add 4 &#181;L of Proteinase K (dissolved in H2O at 20 mg/mL and stored at -20 &#176;C) to each tube and incubate at 45 &#176;C for 1-2 h.</li>
</ol>
7. Purification and recovery of DNA
<ol>
	<li>Add 550 &#181;L of phenol/chloroform/isoamyl alcohol mixture (ratio of 25:24:1) to the centrifuge tube.</li>
	<li>Thoroughly vortex the mixture for 1 min.</li>
	<li>Centrifuge for 15 min at 10,000 x g and aspirate the supernatant.</li>
	<li>Transfer the extracted supernatant from the previous step to a new 1.5 mL centrifuge tube.</li>
	<li>Add 1/10 volume of 3 M sodium acetate solution, 2.5 volumes of absolute ethanol, and 3 &#181;L of glycogen (20 mg/mL) to the tube.</li>
	<li>Place the sample in a refrigerator at -20 &#176;C overnight for precipitation.</li>
	<li>Centrifuge for 15 min at 10,000 x g and 4 &#176;C.</li>
	<li>Discard the supernatant after centrifugation. Wash the pellet three times with 1 mL of 75% ethanol (needs to be prepared fresh) at 10,000 x g.</li>
	<li>Place the washed precipitate on a clean bench to let the alcohol dry.</li>
	<li>Add 50 &#181;L of sterile deionized H2O to sufficiently dissolve the precipitate.</li>
	<li>Ligate the sequencing adaptor to the DNA fragment and use a high-throughput sequencing platform to sequence the DNA.</li>
</ol>
8. DNA repair and Solexa library construction
<ol>
	<li>Repair the DNA ends to generate blunt-ended DNA using a DNA end-repair kit (1-34 &#181;L DNA, 5 &#181;L of 10x end-repair buffer, 5 &#181;L of 2.5 mM each dNTP, 5 &#181;L of 10 mM ATP, 1 &#181;L of end-repair enzyme mix, and H2O to adjust the reaction volume to 49 &#181;L).</li>
	<li>Use a PCR purification kit or phenol: chloroform extraction to purify the DNA. Elute or resuspend the DNA in 30 &#181;L of 1x TE pH 7.4.</li>
	<li>Add &#34;A&#34; to 3&#39; ends (30 &#181;L of DNA from step 2, 2 &#181;L of H2O, 5 &#181;L of 10x Taq buffer, 10 &#181;L of 1 mM dATP, and 3 &#181;L of Taq DNA polymerase). Add the reagents to a 0.2 mL PCR centrifuge tube, mix well, and react in a PCR machine at 72 &#176;C for 10 min.</li>
	<li>Perform linker ligation by mixing 10 &#181;L of DNA, 9.9 &#181;L of H2O, 2.5 &#181;L of T4 DNA ligase buffer, 0.1 &#181;L of adapter oligo mix, and 2.5 &#181;L of T4 DNA ligase. Add the reagents to a 0.2 mL PCR centrifuge tube and mix well. Incubate them at 16 &#176;C for 4 h.</li>
	<li>Purify the DNA using a PCR purification kit according to manufacturer&#39;s protocol. Elute with 20-25 &#181;L of elution buffer.</li>
	<li>Before the DNA library is established, identify the concentration of the purified DNA to confirm its usage for the subsequent sequencing experiments.</li>
	<li>Detect the DNA concentration using a fluorometer. After melting the sample on ice, mix it thoroughly and centrifuge for 30 s at 1000 x g and 4 &#176;C. Then take an appropriate amount of sample and measure it in a fluorometer with a wavelength of 260 nm.</li>
	<li>Place DNA samples with qualified quality and concentration on the Illumina sequencing platform for sequencing.</li>
	<li>Before sequencing, amplify the DNA using PCR primers, PE1.0 and PE2.0, and 2x high fidelity master mix (10.5 &#181;L of DNA, 12.5 &#181;L of 2x high fidelity master mix, 1 &#181;L of PCR primer PE1.0, and 1 &#181;L of PCR primer PE2.0). Add the reagents to a 0.2 mL PCR centrifuge tube and mix well.</li>
	<li>Run the PCR reaction in the PCR machine: 95 &#176;C predenaturation for 2 min; then 35 cycles of 95 &#176;C denaturation for 10 s, annealing at 60 &#176;C for 15 s, extension at 72 &#176;C for 5 s; a final extension at 72 &#176;C for 5 min. Finally, incubate the reaction at 4 &#176;C.</li>
	<li>Use the DNA for cluster generation and perform sequencing-by-synthesis on an Illumina Hiseq 2000. 
	NOTE: In this protocol, Illumina flow cells were used for cluster generation. The sequencing-by-synthesis was performed on an Illumina Genome Analyzer following the manufacturer&#39;s instructions.</li>
</ol>

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The authors have declared that no competing interests exist.

Recently, ChIP-seq has become a widely used genomic analysis method for determining the binding sites of TFs or enrichment sites modified by specific histones. Compared to previous ChIP-seq technology, new ChIP-seq technology is highly sensitive and flexible. Results are provided in high resolution without negative effects, such as the noise signal caused by the non-specific hybridization of nucleic acids. Although this is a common gene expression analysis, many computational methods have been validated, and the complexi...

Epigenetics is a branch of genetic research that refers to the heritable change of gene expression without changing the nucleotide sequence of genes. An increasing number of studies have shown that epigenetic regulation plays an important role in the growth and development of eukaryotic cells, including chromatin that regulates and affects gene expression through the dynamic process of folding and assembly into higher-order structures1,2. Chromatin remodeling and covalent histone modification affect and regulate the function and structure of chromatin through the variation of chromatin polymers, thereby achieving the function of regulating gene expression3,4,5,6. Posttranslational modifications of histone include acetylation, phosphorylation, methylation, monoubiquitination, sumoylation, and ADP ribosylation7,8,9. Histone H3K4 methylation, particularly trimethylation, has been mapped to transcription start sites where it is associated with transcription replication, recombination, repair, and RNA processing in eukaryotes10,11.
ChIP-seq technology was introduced in 2007 and has become the experimental standard for the genome-wide analysis of transcriptional regulation and epigenetic mechanisms12,13. This method is suitable at the genome-wide scale and for obtaining histone or transcription factor interaction information, including DNA segments of DNA binding proteins. Any DNA sequences crosslinked to proteins of interest will coprecipitate as a part of the chromatin complex. New-generation sequencing techniques are also used to sequence 36-100 bp of DNA, which are then matched to the corresponding target genome.
In phytopathogenic fungi, research has recently begun to study how histone methylation modifications regulate their target genes in the process of pathogenicity. Some previous studies proved that the regulation of histone methylase-related genes is mainly reflected in gene silencing and catalyzing the production of Secondary Metabolites (SM). MoSet1 is the H3K4 methylase in M. oryzae. Knockout of this gene results in the complete deletion of H3K4me3 modification14. Compared with the wild-type strain, the expression of the gene MoCEL7C in the mutant is inhibited in the CMC-induced state and in the non-induced state (glucose or cellobiose), the expression of MoCEL7C increased15. In Fusarium graminearum, KMT6 can catalyze the methylation modification of H3K27me3, regulate the normal development of fungi, and help regulate the &#34;cryptic genome&#34; containing the SM gene cluster16,17,18,19. In 2013, Connolly reported that H3K9 and H3K27 methylation regulates the pathogenic process of fungi through secondary metabolites and effector factors that regulate the inhibition of target genes20. In Aspergillus, the modification of histones H3K4me2 and H3K4me3 is related to gene activation and plays an important role in controlling the chromatin level regulation of SM gene clusters21. In M. oryzae, Tig1 (homologous to Tig1 in yeast and mammals) is an HADC (histone deacetylase)22. Knockout of the Tig1 gene leads to the complete loss of pathogenicity and spore production ability in the null mutant. It is more sensitive to a peroxygen environment, which cannot produce infective hyphae22.
The rice blast caused by M. oryzae. is one of the most serious rice diseases in most rice-growing areas in the world19. Due to its representative infection process, M. oryzae is similar to the infection process of many important pathogenic fungi. As it can easily carry out molecular genetic operations, the fungus has become a model organism for studying fungal-plant interactions23. Blocking every step of the infection process of M. oryzae may result in unsuccessful infection. The morphological changes during the infection process are strictly regulated by the entire genome function and gene transcription. Among them, epigenetic modifications such as histone methylation play an essential role in the transcriptional regulation of functional genes24,25. However, so far, few studies have been done on the molecular mechanism of epigenetic modifications such as histone methylation and histone acetylation in the transcription of pathogenesis genes in M. oryzae. Therefore, further developing the epigenetic regulation mechanism of the rice blast fungus while researchinh the upstream and downstream regulatory network of these pathogenic related genes will help develop rice blast prevention and control strategies.
With the development of functional genomics such as ChIP-seq, especially in epigenetics, this high-throughput data acquisition method has accelerated research on chromosomes. Using the ChIP-seq experimental technology, the genome-wide distribution of histone methylation (such as H3K4me3, H3K27me3, H3K9me3) in M. oryzae and other filamentous fungi can be identified. Therefore, this method can help elucidate the molecular mechanisms underlying how epigenetic modifications regulate their candidate target genes during fungal pathogenesis in plant pathology.

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			<td>acidic casein hydrolysate</td>
			<td>WAKO</td>
			<td>65072-00-6</td>
			<td>Medium configuration</td>
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		<tr>
			<td>agar powder</td>
			<td>scientan</td>
			<td>9002-18-0</td>
			<td>Medium configuration</td>
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		<tr>
			<td>deoxycholic acid</td>
			<td>MedChemExpress</td>
			<td>83-44-3</td>
			<td>protein and dissolution</td>
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		<tr>
			<td>DNA End-Repair kit</td>
			<td>NovoBiotec</td>
			<td>ER81050</td>
			<td>Repair DNA or cDNA damaged by enzymatic or mechanical shearing</td>
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		<tr>
			<td>Dynabeads</td>
			<td>Invitrogen</td>
			<td>no.100.02D</td>
			<td>Binding target</td>
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		<tr>
			<td>EB buffer</td>
			<td>JIMEI</td>
			<td>JC2174</td>
			<td>Membrane and liquid</td>
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		<tr>
			<td>EDTA</td>
			<td>ThermoFisher</td>
			<td>AM9912</td>
			<td>protease inhibitor</td>
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			<td>enzymatic casein hydrolysate</td>
			<td>Sigma</td>
			<td>91079-40-2</td>
			<td>Medium configuration</td>
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			<td>glucose</td>
			<td>Sigma</td>
			<td>50-99-7</td>
			<td>Medium configuration</td>
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			<td>glycogen</td>
			<td>ThermoFisher</td>
			<td>AM9510</td>
			<td>Precipitant action</td>
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			<td>H3K4me3 antibodies</td>
			<td>Abcam</td>
			<td>ab8580</td>
			<td>Immune response to H3K4me3 protein</td>
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		<tr>
			<td>illumina Genome Analyzer</td>
			<td>illumina</td>
			<td>illumina Hiseq 2000</td>
			<td>Large configuration</td>
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		<tr>
			<td>Illumina PCR primers</td>
			<td>illumina</td>
			<td>CleanPlex</td>
			<td>Random universal primer</td>
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			<td>isoamyl alcohol</td>
			<td>chemical book</td>
			<td>30899-19-5</td>
			<td>Purified DNA</td>
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			<td>LiCl</td>
			<td>ThermoFisher</td>
			<td>AM9480</td>
			<td>specific removal RNA</td>
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		<tr>
			<td>lysing enzymes</td>
			<td>Sigma</td>
			<td>L1412-10G</td>
			<td>cell lysis buffer</td>
		</tr>
		<tr>
			<td>Mouse IgG</td>
			<td>Yeasen</td>
			<td>36111ES10</td>
			<td>Animal normal immunoglobulin</td>
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		<tr>
			<td>NaCl solution</td>
			<td>ThermoFisher</td>
			<td>7647-14-5</td>
			<td>Medium configuration</td>
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			<td>NaHCO3</td>
			<td>Seebio</td>
			<td>SH30173.08*</td>
			<td>preparation of protein complex eluent</td>
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			<td>NP-40</td>
			<td>ThermoFisher</td>
			<td>85124</td>
			<td>cell lysate to promote cell lysis</td>
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			<td>PCR Purification kit</td>
			<td>Qiagen</td>
			<td>28004</td>
			<td>The purification procedure removes primers from DNA samples</td>
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			<td>protease inhibitors</td>
			<td>ThermoFisher</td>
			<td>A32965</td>
			<td>A protein inhibitor that decreases protein activity</td>
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			<td>Proteinase K</td>
			<td>ThermoFisher</td>
			<td>AM2546</td>
			<td>DNA Extraction Reagent</td>
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		<tr>
			<td>Qubit 4.0</td>
			<td>ThermoFisher</td>
			<td>Q33226</td>
			<td>Medium configuration</td>
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			<td>RIPA buffer</td>
			<td>ThermoFisher</td>
			<td>9806S</td>
			<td>cell lysis buffer</td>
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			<td>RNase A</td>
			<td>ThermoFisher</td>
			<td>AM2271</td>
			<td>Purified DNA</td>
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			<td>SDS</td>
			<td>ThermoFisher</td>
			<td>AM9820</td>
			<td>cover up the charge differences</td>
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			<td>sodium acetate solution</td>
			<td>ThermoFisher</td>
			<td>R1181</td>
			<td>Acetic acid buffer</td>
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			<td>sodium deoxycholate</td>
			<td>ThermoFisher</td>
			<td>89904</td>
			<td>inhibition of protease degradation</td>
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			<td>T4 DNA ligase</td>
			<td>ThermoFisher</td>
			<td>EL0011</td>
			<td>Under the condition of ATP as coenzyme, DNA ligase</td>
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		<tr>
			<td>T4 DNA ligase buffer</td>
			<td>ThermoFisher</td>
			<td>B69</td>
			<td>DNA ligase buffer</td>
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			<td>Tris-HCl</td>
			<td>ThermoFisher</td>
			<td>1185-53-1</td>
			<td>buffer action</td>
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		<tr>
			<td>Triton X-100</td>
			<td>ThermoFisher</td>
			<td>HFH10</td>
			<td>keep the membrane protein stable</td>
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		<tr>
			<td>yeast extract</td>
			<td>OXOID</td>
			<td>LP0021</td>
			<td>Medium configuration</td>
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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.

The whole flow chart of the ChIP-seq method is shown in Figure 1. ChIP-seq experiments were performed using antibodies against H3K4me3 in the wild-type strain P131 and three null mutant strains that were devoid of mobre2, mospp1, and moswd2 gene to verify the whole genome-wide profile of histone H3K4me3 distribution in M. oryzae. The protoplasts of the wild-type strain, &#916;mobre2, &#916;mospp1, and &#916;moswd2, were prepared ...

Watch this Scientific Journal Video about Genome-wide Analysis of Histone Modifications Distribution using the Chromatin Immunoprecipitation Sequencing Method in Magnaporthe oryzae at JoVE.com

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.

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 ...

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2.8K Views.  Beijing University of Agriculture. 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.

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

Genome-wide Determination of Mammalian Replication Timing by DNA Content Measurement

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic region is replicated at a distinct time during S phase through the simultaneous activation of multiple origins of replication. Time of replication (ToR) correlates with many genomic and epigenetic features and is linked to mutation rates and cancer. Comprehending the full genomic view of the replication program, in health and disease is a major future goal and challenge.
This article describes in detail the &#34;Copy Number Ratio of S/G1 for mapping genomic Time of Replication&#34; method (herein called: CNR-ToR), a simple approach to map the genome wide ToR of mammalian cells. The method is based on the copy number differences between S phase cells and G1 phase cells. The CNR-ToR method is performed in 6 steps: 1. Preparation of cells and staining with propidium iodide (PI); 2. Sorting G1 and S phase cells using fluorescence-activated cell sorting (FACS); 3. DNA purification; 4. Sonication; 5. Library preparation and sequencing; and 6. Bioinformatic analysis. The CNR-ToR method is a fast and easy approach that results in detailed replication maps.

We describe here a relatively fast and simple approach for mapping genome-wide mammalian replication timing, from cell isolation to the basic analysis of the sequencing results. A genomic map of a representative replication program will be provided following the protocol.

Replication of the genome occurs during S phase of the cell cycle in a highly regulated process that ensures the fidelity of DNA duplication. Each genomic ...

Analysis of the c-KIT Ligand Promoter Using Chromatin Immunoprecipitation

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. Therefore, DNA-protein interactions regulate multiple physiological, pathophysiological, and biological functions, such as cell differentiation, cell proliferation, cell cycle control, chromosome stability, epigenetic gene regulation, and cell transformation. In eukaryotic cells, the DNA interacts with histone and nonhistone proteins and is condensed into chromatin. Several technical tools can be used to analyze DNA-protein interactions, such as the Electrophoresis (gel) Mobility Shift Assay (EMSA) and DNase I footprinting. However, these techniques analyze the protein-DNA interaction in vitro, not within the cellular context. Chromatin immunoprecipitation (ChIP) is a technique that captures proteins at their specific DNA binding sites, thereby allowing for the identification of DNA-protein interactions within their chromatin context. It is done by fixation&#160;of the DNA-protein interaction, followed by&#160;immunoprecipitation of the protein of interest. Subsequently, the genomic site that the protein was bound to is characterized. Here, we describe and discuss ChIP and demonstrate its analytical value for the identification of the Transforming Growth Factor-&#946; (TGF-&#946;)-induced binding of the transcription factor SMAD2 to SMAD Binding Elements (SBE) within the promoter region of the tyrosine-protein kinase Kit (c-KIT) receptor ligand Stem Cell Factor (SCF).

DNA-protein interactions are essential for multiple biological processes. During the evaluation of cellular functions, the analysis of DNA-protein interactions is indispensable for understanding gene regulation. Chromatin immunoprecipitation (ChIP) is a powerful tool to analyze such interactions in vivo.

Multiple cellular processes, including DNA replication and repair, DNA recombination, and gene expression, require interactions between proteins and DNA. ...

Chromatin Immunoprecipitation (ChIP) in Mouse T-cell Lines

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational modifications (PTMs) of histone tails, the exchange of canonical histones with histone variants, and nucleosome eviction. Such regulation requires the binding of signal-sensitive transcription factors (TFs) that recruit chromatin-modifying enzymes at regulatory elements defined as enhancers. Understanding how signaling cascades regulate enhancer activity requires a comprehensive analysis of the binding of TFs, chromatin modifying enzymes, and the occupancy of specific histone marks and histone variants. Chromatin immunoprecipitation (ChIP) assays utilize highly specific antibodies to immunoprecipitate specific protein/DNA complexes. The subsequent analysis of the purified DNA allows for the identification the region occupied by the protein recognized by the antibody. This work describes a protocol to efficiently perform ChIP of histone proteins in a mature mouse T-cell line. The presented protocol allows for the performance of ChIP assays in a reasonable timeframe and with high reproducibility.

Chromatin Immunoprecipitation ChIP in Mouse T-cell Lines

This work describes a protocol for chromatin immunoprecipitation (ChIP) using a mature mouse T-cell line. This protocol is suitable to investigate the distribution of specific histone marks at specific promoter sites or genome-wide.

Signaling pathways regulate gene expression programs via the modulation of the chromatin structure at different levels, such as by post-translational ...

Generation of Native Chromatin Immunoprecipitation Sequencing Libraries for Nucleosome Density Analysis

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution based analysis methodology (nucleosome density ChIP-seq; ndChIP-seq) that enables the generation of combined measurements of micrococcal nuclease (MNase) accessibility with histone modification genome-wide. Nucleosome position and local density, and the posttranslational modification of their histone subunits, act in concert to regulate local transcription states. Combinatorial measurements of nucleosome accessibility with histone modification generated by ndChIP-seq allows for the simultaneous interrogation of these features. The ndChIP-seq methodology is applicable to small numbers of primary cells inaccessible to cross-linking based ChIP-seq protocols. Taken together, ndChIP-seq enables the measurement of histone modification in combination with local nucleosome density to obtain new insights into shared mechanisms that regulate RNA transcription within rare primary cell populations.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) methodology for the generation of sequence datasets suitable for a nucleosome density ChIP-seq analytical framework integrating micrococcal nuclease (MNase) accessibility with histone modification measurements.

We present a modified native chromatin immunoprecipitation sequencing (ChIP-seq) experimental protocol compatible with a Gaussian mixture distribution ...

Chromatin Immunoprecipitation (ChIP) Protocol for Low-abundance Embryonic Samples

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, transcription factors, or chromatin-modifying enzymes at a given locus or on a genome-wide scale. The combination of ChIP assays with next-generation sequencing (i.e., ChIP-Seq) is a powerful approach to globally uncover gene regulatory networks and to improve the functional annotation of genomes, especially of non-coding regulatory sequences. ChIP protocols normally require large amounts of cellular material, thus precluding the applicability of this method to investigating rare cell types or small tissue biopsies. In order to make the ChIP assay compatible with the amount of biological material that can typically be obtained in vivo during early vertebrate embryogenesis, we describe here a simplified ChIP protocol in which the number of steps required to complete the assay were reduced to minimize sample loss. This ChIP protocol has been successfully used to investigate different histone modifications in various embryonic chicken and adult mouse tissues using low to medium cell numbers (5 x 104 - 5 x 105 cells). Importantly, this protocol is compatible with ChIP-seq technology using standard library preparation methods, thus providing global epigenomic maps in highly relevant embryonic tissues.

Chromatin Immunoprecipitation ChIP Protocol for Low-abundance Embryonic Samples

Here, we describe a chromatin immunoprecipitation (ChIP) and ChIP-seq library preparation protocol to generate global epigenomic profiles from low-abundance chicken embryonic samples.

Chromatin immunoprecipitation (ChIP) is a widely-used technique for mapping the localization of post-translationally modified histones, histone variants, ...

Mapping Genome-wide Accessible Chromatin in Primary Human T Lymphocytes by ATAC-Seq

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions of chromatin. These regions represent regulatory DNA elements (e.g., promoters, enhancers, locus control regions, insulators) to which transcription factors bind. Mapping the accessible chromatin landscape is a powerful approach for uncovering active regulatory elements across the genome. This information serves as an unbiased approach for discovering the network of relevant transcription factors and mechanisms of chromatin structure that govern gene expression programs. ATAC-seq is a robust and sensitive alternative to DNase I hypersensitivity analysis coupled with next-generation sequencing (DNase-seq) and formaldehyde-assisted isolation of regulatory elements (FAIRE-seq) for genome-wide analysis of chromatin accessibility and to the sequencing of micrococcal nuclease-sensitive sites (MNase-seq) to determine nucleosome positioning. We present a detailed ATAC-seq protocol optimized for human primary immune cells i.e. CD4+ lymphocytes (T helper 1 (Th1) and Th2 cells). This comprehensive protocol begins with cell harvest, then describes the molecular procedure of chromatin tagmentation, sample preparation for next-generation sequencing, and also includes methods and considerations for the computational analyses used to interpret the results. Moreover, to save time and money, we introduced quality control measures to assess the ATAC-seq library prior to sequencing. Importantly, the principles presented in this protocol allow its adaptation to other human immune and non-immune primary cells and cell lines. These guidelines will also be useful for laboratories which are not proficient with next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin coupled with high-throughput sequencing (ATAC-seq) is a genome-wide method to uncover accessible chromatin. This is a step-by-step ATAC-seq protocol, from molecular to the final computational analysis, optimized for human lymphocytes (Th1/Th2). This protocol can be adopted by researchers without prior experience in next-generation sequencing methods.

Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq) is a method used for the identification of open (accessible) regions ...

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a protocol for performing the chromatin conformation capture technique in situ Hi-C on staged Drosophila melanogaster embryo populations. The result is a sequencing library that allows the mapping of all chromatin interactions that occur in the nucleus in a single experiment. Embryo sorting is done manually using a fluorescent stereo microscope and a transgenic fly line containing a nuclear marker. Using this technique, embryo populations from each nuclear division cycle, and with defined cell cycle status, can be obtained with very high purity. The protocol may also be adapted to sort older embryos beyond gastrulation. Sorted embryos are used as inputs for in situ Hi-C. All experiments, including sequencing library preparation, can be completed in five days. The protocol has low input requirements and works reliably using 20 blastoderm stage embryos as input material. The end result is a sequencing library for next generation sequencing. After sequencing, the data can be processed into genome-wide chromatin interaction maps that can be analyzed using a wide range of available tools to gain information about topologically associating domain (TAD) structure, chromatin loops, and chromatin compartments during Drosophila development.

Generation of Genome-wide Chromatin Conformation Capture Libraries from Tightly Staged Early Drosophila Embryos

This work describes a protocol for the generation of high resolution in situ Hi-C libraries from tightly staged pre-gastrulation Drosophila melanogaster embryos.

Investigating the three-dimensional architecture of chromatin offers invaluable insight into the mechanisms of gene regulation. Here, we describe a ...

Chromatin Immunoprecipitation (ChIP) of Histone Modifications from Saccharomyces cerevisiae

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes that add or remove these marks in response to signals received by the cell. These PTMS are key contributors to the regulation of processes such as gene expression control and DNA repair. Chromatin immunoprecipitation (chIP) has been an instrumental approach for dissecting the abundance and localization of many histone PTMs throughout the genome in response to diverse perturbations to the cell. Here, a versatile method for performing chIP of post-translationally modified histones from the budding yeast Saccharomyces cerevisiae (S. cerevisiae) is described. This method relies on crosslinking of proteins and DNA using formaldehyde treatment of yeast cultures, generation of yeast lysates by bead beating, solubilization of chromatin fragments by micrococcal nuclease, and immunoprecipitation of histone-DNA complexes. DNA associated with the histone mark of interest is purified and subjected to quantitative PCR analysis to evaluate its enrichment at multiple loci throughout the genome. Representative experiments probing the localization of the histone marks H3K4me2 and H4K16ac in wildtype and mutant yeast are discussed to demonstrate data analysis and interpretation. This method is suitable for a variety of histone PTMs and can be performed with different mutant strains or in the presence of diverse environmental stresses, making it an excellent tool for investigating changes in chromatin dynamics under different conditions.

Chromatin Immunoprecipitation ChIP of Histone Modifications from Saccharomyces cerevisiae

Here, we describe a protocol for chromatin immunoprecipitation of modified histones from the budding yeast Saccharomyces cerevisiae. Immunoprecipitated DNA is subsequently used for quantitative PCR to interrogate the abundance and localization of histone post-translational modifications throughout the genome.

Histone post-translational modifications (PTMs), such as acetylation, methylation and phosphorylation, are dynamically regulated by a series of enzymes ...

Promoter Capture Hi-C: High-resolution, Genome-wide Profiling of Promoter Interactions

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the spatio-temporal expression of their target genes through physical contact, often bridging considerable (in some cases hundreds of kilobases) genomic distances and bypassing nearby genes. The human genome harbors an estimated one million enhancers, the vast majority of which have unknown gene targets. Assigning distal regulatory regions to their target genes is thus crucial to understand gene expression control. We developed Promoter Capture Hi-C (PCHi-C) to enable the genome-wide detection of distal promoter-interacting regions (PIRs), for all promoters in a single experiment. In PCHi-C, highly complex Hi-C libraries are specifically enriched for promoter sequences through in-solution hybrid selection with thousands of biotinylated RNA baits complementary to the ends of all promoter-containing restriction fragments. The aim is to then pull-down promoter sequences and their frequent interaction partners such as enhancers and other potential regulatory elements. After high-throughput paired-end sequencing, a statistical test is applied to each promoter-ligated restriction fragment to identify significant PIRs at the restriction fragment level. We have used PCHi-C to generate an atlas of long-range promoter interactions in dozens of human and mouse cell types. These promoter interactome maps have contributed to a greater understanding of mammalian gene expression control by assigning putative regulatory regions to their target genes and revealing preferential spatial promoter-promoter interaction networks. This information also has high relevance to understanding human genetic disease and the identification of potential disease genes, by linking non-coding disease-associated sequence variants in or near control sequences to their target genes.

DNA regulatory elements, such as enhancers, control gene expression by physically contacting target gene promoters, often through long-range chromosomal interactions spanning large genomic distances. Promoter Capture Hi-C (PCHi-C) identifies significant interactions between promoters and distal regions, enabling the assignment of potential regulatory sequences to their target genes.

The three-dimensional organization of the genome is linked to its function. For example, regulatory elements such as transcriptional enhancers control the ...

Genome-wide Surveillance of Transcription Errors in Eukaryotic Organisms

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that control the fidelity of transcription. To fill this gap in scientific knowledge, we recently optimized the circle-sequencing assay to detect transcription errors throughout the transcriptome of Saccharomyces cerevisiae, Drosophila melanogaster, and Caenorhabditis elegans. This protocol will provide researchers with a powerful new tool to map the landscape of transcription errors in eukaryotic cells so that the mechanisms that control the fidelity of transcription can be elucidated in unprecedented detail.

This protocol provides researchers with a new tool to monitor the fidelity of transcription in multiple model organisms.

Accurate transcription is required for the faithful expression of genetic information. Surprisingly though, little is known about the mechanisms that ...