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

Summary

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.

Abstract

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.

Introduction

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 structures^1,2. 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 expression^3,4,5,6. Posttranslational modifications of histone include acetylation, phosphorylation, methylation, monoubiquitination, sumoylation, and ADP ribosylation^7,8,9. 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 eukaryotes^10,11.

ChIP-seq technology was introduced in 2007 and has become the experimental standard for the genome-wide analysis of transcriptional regulation and epigenetic mechanisms^12,13. 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 "cryptic genome" containing the SM gene cluster^16,17,18,19. 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 genes^24,25. 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.

Protocol

1. Preparation of protoplasts from M. oryzae

1. Prepare the oatmeal-tomato agar (OTA).
   1. 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.
   2. Pick ripe tomatoes and peel them. Squeeze the juice, and filter through two layers of gauze to collect 150 mL of the filtered juice.
   3. Mix all the tomato juice and the prepared oat filtrate thoroughly and add ddH₂O up to 1000 mL.
   4. 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 °C.
2. 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 °C.
3. Use a sterilized toothpick to dig out a small piece of mycelium from M. oryzae (the wild-type strain P131, knockout strains Δmobre2, Δmospp1, and Δmoswd2) and place them on the prepared OTA dishes. Culture them for 4-6 days at 28 °C under light conditions.
   NOTE: Turn the Petri dish upside down to prevent pollution.
4. Add 1000 µL 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 µL pipette.
   NOTE: The hyphae grew on the OTA dishes for 4-6 days.
5. Scrape the mycelia of the wild-type strain and knockout strain with an inoculation loop.
6. Collect the mycelia debris and transfer them to 250 mL of liquid Complete Medium (CM).
7. Grow the fungal debris in a triangular flask at 28 °C for 36 h with shaking at 150 rpm.
8. Use a funnel to filter and collect the fungal hyphae.
9. Wash the fungal hyphae with 500 mL of 0.7 M NaCl solution.
10. Collect the fungal mycelium and weigh it.
    NOTE: The mycelium does not need to be dried before weighing.
11. Add ~1 mL of lysis enzyme permeation solution per 1 g of the fungal mycelium.
    1. Prepare the 20 mg/mL lysis enzyme permeation solution by dissolving the lysis enzyme from Trichoderma harzianum in 0.7 M NaCl.
12. Place the hyphae for lysing at 28 °C for 3-4 h with shaking at 150 rpm.
13. Wash the lysed hyphae with 50 mL of 0.7 M NaCl solution.
14. Collect the protoplasts and centrifuge for 15 min at 2,000 x g and 4 °C.
15. After centrifugation, discard the supernatant carefully. Resuspend the protoplasts in 20 mL of 0.7 M NaCI buffer at 4 °C.

2. In vivo crosslinking and sonication

1. Add 55 µL of 37% formaldehyde (add formaldehyde drop by drop until the final concentration is 1%) to 2 mL of NaCI buffer containing protoplast for crosslinking.
2. Incubate the protoplasts at25 °Cfor 10 min.
3. Add 20 µL of 10x glycine to each tube to quench the unreacted formaldehyde.
4. Swirl to mix and incubate at 25 °C for 5 min.
5. Centrifuge for 15 min at 2,000 x g and 4 °C.
6. After centrifugation, discard the supernatant carefully. Resuspend the pellet in 1 mL of 0.7 M NaCI solution.
7. Centrifuge for 10 min at 2,000 x g and 4 °C.
8. After centrifugation, discard the supernatant carefully. Resuspend the pellet in 750 µL 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).
9. Add 37.5 µL of 20x protease inhibitor.
10. Transfer the protoplasts from the previous step to a 1.5 mL centrifuge tube.
11. Perform sonication (25% W, output 3 s, stop 5 s, 4 °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 °C at this step.
12. If optimal conditions for sonication have already been determined, proceed to the next step.
13. If desired, remove 5 µL of protoplasts for agarose gel analysis (unsheared DNA).
14. Shear the chromatin by sonication with an ultrasonic homogenizer for 8 min (25% W, output 3 s, stop 5 s, 4 °C).
15. After the sample is ultrasonically broken, take out a part of the sample as "input".. The input does not perform the ChIP experiment and contains all DNA and protein released after the sample is sonicated.
16. 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).
17. Place the sonicated tube on ice to prevent protein degradation.
18. Centrifuge for 10 min at 10,000 x g and 4 °C.
19. After centrifugation, transfer the centrifuged supernatant to a new 1.5 mL centrifuge tube and store it at -80 °C for later use. The chromatin solution obtained in this step can be used for subsequent IP.
20. Before performing the IP experiment, dilute each chromatin sample to a ratio of 1:10 with 1x RIPA buffer (e.g., add 10 µL of chromatin sample to 1 µL of 1×RIPA buffer).

3. IP of crosslinked protein/DNA

1. Pipette 50 µL 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.
2. 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 µL of 1x RIPA buffer to each tube.
3. Add 300 µL of chormatin sample (2 x 10⁷ cells were used), 100 µL of superparamagnetic protein beads and 4 µL of H3K4me3 antibody to the tube.
4. 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 °C (see Table of Materials).
5. After mixing well, place the samples on a rotary shaker and incubate overnight at 4 °C, 30 x g.
   ​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.

4. Collecting and rinsing the IP products

1. Pellet the superparamagnetic protein beads by placing them on a magnetic stand. Aspirate and discard the supernatant.
2. Wash the superparamagnetic protein bead-antibody/chromatin complex by resuspending the beads in 1 mL of 1x RIPA buffer.
3. Rinse the tube on a rotary shaker for 5 min and remove the supernatant at 30 x g.
4. 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).
5. Rinse the tube on a rotary shaker for 5 min and remove the supernatant.
6. 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.
7. Place the tube on a rotary shaker for 5 min and remove the supernatant.
8. 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.
9. Place the tube on a rotary shaker for 5 min and remove the supernatant.
10. 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.
11. Add 1 mL of TE buffer (10 mM Tris-HCl pH 8.1, 1 mM EDTA).
12. Place the tube on a rotary shaker for 5 min at 30 x g.
13. Wash the tube again with 1 mL TE buffer and remove the supernatant with a pipette.
14. Collect the beads.

5. Elution of protein/DNA complexes

1. Prepare the elution buffer (10 µL of 20% SDS, 20 µL of 1 M NaHCO₃, 170 µL of sterile distilled H₂O) for all the IP and input tubes.
2. Add 100 µL of elution buffer to each centrifuge tube.
3. Elution at 65 °C for 15 min.
4. Centrifuge for 1 min at 10,000 x g and 4 °C and collect the supernatant into new centrifuge tubes.
5. Repeat steps 5.2 - 5.4 and combine the eluates. Add 190 µL of elution buffer to the 10 µL of the input DNA. (total volume = 200 µL).

6. Reverse crosslinking of protein/DNA complexes

1. Add 8 µL of 5 M NaCl to all the tubes and incubate at 65 °C for 4-5 h or overnight to reverse the DNA-protein crosslinks.
   NOTE: After this step, store the samples at -20 °C, and continue the protocol the following day, if necessary.
2. Add 1 µL of RNase A and incubate for 30 min at 37 °C.
3. Add 4 µL of Proteinase K (dissolved in H₂O at 20 mg/mL and stored at -20 °C) to each tube and incubate at 45 °C for 1-2 h.

7. Purification and recovery of DNA

1. Add 550 µL of phenol/chloroform/isoamyl alcohol mixture (ratio of 25:24:1) to the centrifuge tube.
2. Thoroughly vortex the mixture for 1 min.
3. Centrifuge for 15 min at 10,000 x g and aspirate the supernatant.
4. Transfer the extracted supernatant from the previous step to a new 1.5 mL centrifuge tube.
5. Add 1/10 volume of 3 M sodium acetate solution, 2.5 volumes of absolute ethanol, and 3 µL of glycogen (20 mg/mL) to the tube.
6. Place the sample in a refrigerator at -20 °C overnight for precipitation.
7. Centrifuge for 15 min at 10,000 x g and 4 °C.
8. 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.
9. Place the washed precipitate on a clean bench to let the alcohol dry.
10. Add 50 µL of sterile deionized H₂O to sufficiently dissolve the precipitate.
11. Ligate the sequencing adaptor to the DNA fragment and use a high-throughput sequencing platform to sequence the DNA.

8. DNA repair and Solexa library construction

1. Repair the DNA ends to generate blunt-ended DNA using a DNA end-repair kit (1-34 µL DNA, 5 µL of 10x end-repair buffer, 5 µL of 2.5 mM each dNTP, 5 µL of 10 mM ATP, 1 µL of end-repair enzyme mix, and H₂O to adjust the reaction volume to 49 µL).
2. Use a PCR purification kit or phenol: chloroform extraction to purify the DNA. Elute or resuspend the DNA in 30 µL of 1x TE pH 7.4.
3. Add "A" to 3' ends (30 µL of DNA from step 2, 2 µL of H₂O, 5 µL of 10x Taq buffer, 10 µL of 1 mM dATP, and 3 µL 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 °C for 10 min.
4. Perform linker ligation by mixing 10 µL of DNA, 9.9 µL of H₂O, 2.5 µL of T4 DNA ligase buffer, 0.1 µL of adapter oligo mix, and 2.5 µL of T4 DNA ligase. Add the reagents to a 0.2 mL PCR centrifuge tube and mix well. Incubate them at 16 °C for 4 h.
5. Purify the DNA using a PCR purification kit according to manufacturer's protocol. Elute with 20-25 µL of elution buffer.
6. Before the DNA library is established, identify the concentration of the purified DNA to confirm its usage for the subsequent sequencing experiments.
7. 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 °C. Then take an appropriate amount of sample and measure it in a fluorometer with a wavelength of 260 nm.
8. Place DNA samples with qualified quality and concentration on the Illumina sequencing platform for sequencing.
9. Before sequencing, amplify the DNA using PCR primers, PE1.0 and PE2.0, and 2x high fidelity master mix (10.5 µL of DNA, 12.5 µL of 2x high fidelity master mix, 1 µL of PCR primer PE1.0, and 1 µL of PCR primer PE2.0). Add the reagents to a 0.2 mL PCR centrifuge tube and mix well.
10. Run the PCR reaction in the PCR machine: 95 °C predenaturation for 2 min; then 35 cycles of 95 °C denaturation for 10 s, annealing at 60 °C for 15 s, extension at 72 °C for 5 s; a final extension at 72 °C for 5 min. Finally, incubate the reaction at 4 °C.
11. 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's instructions.

Representative Results

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, Δmobre2, Δmospp1, and Δmoswd2, were prepared and sonicated at 25% W, output 3 s, stop 5 s, at 4 °C. Further, the chromatin was immunopurified with H3K4me3 antibody and Dynabeads protein A/G. Subsequently, DNA fragments were extracted using the phenol-chloroform method for constructing a sequencing library and sequenced with single ends on a high-throughput sequencing platform.

The representative results of the wild-type, Δmobre2, Δmospp1, and Δmoswd2 strains with ChIP-seq method using the H3K4me3 antibody are shown in Figure 2. The H3K4me3 signals of the Δmobre2, Δmospp1, and Δmoswd2 deletion mutants were significantly decreased at its functional target regions. As shown in Figure 2, some selected candidate target genes, including MGG_14897, MGG_04237, MGG_04236, and MGG_04235, were mapped for H3K4me3 distribution. Compared to the wild-type strain P131, the signals of enriched H3K4me3-ChIP-seq reads in the Δmobre2, Δmospp1, and Δmoswd2 deletion mutants were largely decreased (Figure 2)²⁶. These results suggest that the H3K4me3 modification plays important roles in regulating target gene expression in M. oryzae.

Figure 1. The flow chart of the ChIP-seq method in M. oryzae. (A) The genomic DNA of M. oryzae wascrosslinked with 1% formaldehyde. (B) Lysed blast fungus cells, broken DNA, free DNA, and histone binding DNA were subsequently obtained. (C) DNA fragments bound to histones and were extracted by specific binding to the H3K4me3 antibody. (D) Through reverse crosslinking, purified DNA subsequently obtained DNA fragments modified by H3K4me3 histones. (E-F) DNA fragments were sequenced, the sequencing results were compared, and sequences were identified in the M. oryzae DNA group. (G) Specific genes and loci of H3K4me3 histones in M. oryzae were retrieved. Please click here to view a larger version of this figure.

Figure 2. The Δmobre2, Δmospp1, and Δmoswd2 deletion mutants significantly decreased H3K4me3 profiles in their target regions. The H3K4me3-ChIP-seq distribution of enriched peaks around the coding regions of overlapped genes in Δmobre2, Δmospp1, and Δmoswd2 deletion mutants are decresased compared to the wild-type strain in the MGG_14897,MGG_04237, MGG_04236 and MGG_04235 genes²⁶. The number in WT (input) labelled as [0-2074] signify means the results of ChIP in the range of genomic DNA [0-2074]. [0-2074] refers to 0-2074bp of Chromosome 6.The figure shows a random selection of the sequencing results, which only represents the DNA distribution on Chromosome 6. The complete sequencing results have been submitted to Genbank. (https//www.ncbi.nlm.nih.gov/bioproject/ accession 649321)²⁶. Please click here to view a larger version of this figure.

Serial number	Sample name		Sample serial number		Number of tubes	Total (μg)	Fragment distribution			Database type	Remarks
1	input		WH1703004169		1	2.7948	The main peak is below 100bp, but there is DNA distribution between 100bp-500bp			ChIP-seq	Fragment is too small
	P131(2)
2	Input		WH1703004170		1	2.4748	The main peak is below 100bp, but there is DNA distribution between 100bp-500bp			ChIP-seq	Fragment is too small
	Δmobre2(3)
3	input Δmospp1(4)		WH1703004171		1	3.22	The main peak is below 100bp, but there is DNA distribution between 100bp-500bp			ChIP-seq	Fragment is too small
4	input Δmoswd(5)		WH1703004172		1	3.97	The main peak is below 100bp, but there is DNA distribution between 100bp-500bp			ChIP-seq	Fragment is too small
5	P131(2)		WH1703004174		1	0.0735	The main peak is between 100bp-500bp			ChIP-seq
6	Δmobre2(3)		WH1703004175		1	0.0491	The main peak is between 100bp-500bp			ChIP-seq
7	Δmospp1(4)		WH1703004176		1	0.0288	The main peak is between 100bp-500bp			ChIP-seq
8	Δmoswd(5)		WH1703004177		1	0.0527	The main peak is between 100bp-500bp			ChIP-seq

Table 1. The total amount of DNA in this experiment. The total amount of input P131(2) is 2.7948 µg, the total amount of Δmobre2(3) (input) is 2.4748 µg, the total amount of Δmospp1(4) (input) is 3.22 µg, the total amount of Δmoswd2 (5) (input) is 3.97 µg, and the total amount of P131(2) is 0.0735 µg, the total amount of Δmobre2(3) is 0.0491µg, the total amount of Δmospp1(4) is 0.0288 µg, the total amount of Δmoswd2(5) is 0.0527 µg.

Figure 3. Electrophoresis detection of DNA after ultrasound. After ultrasound sonication, the DNA is subjected to a 1% agarose gel experiment to analyze the length of DNA fragments. The sonicated DNA fragment length is from 200-500 bp, and these DNA fragments can be used for the following steps of ChIP-seq. Please click here to view a larger version of this figure.

Figure 4. Bioanalyzer trace of Input and ChIP samples. The figure shows the fragment distribution of each sample, where the abscissa represents the fragment size, and the ordinate represents the peak size. The samples running on the bioanalyzer are input P131(2), Δmobre2(3) (input), Δmospp1(4) (input), Δmoswd2(5) (input), P131(2), Δmobre2(3), Δmospp1(4), Δmoswd(5). Among them, the distribution of input P131(2), Δmobre2(3) (input), Δmospp1(4) (input), Δmoswd2(5) (input) shows the main peak is below 100 bp, but there is DNA distribution at 100-500 bp. The true distribution of P131(2), Δmobre2(3), Δmospp1(4), and Δmoswd2(5) is between 100-500 bp as the main peak²⁶. Please click here to view a larger version of this figure.

Discussion

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 complexity of ChIP-seq data in terms of noise and variability makes this problem particularly difficult for ChIP-seq to overcome. In terms of data analysis, managing and analyzing the large amount of data generated by ChIP-seq experiments is also a challenge that has yet to be adequately addressed.

There are several key steps in the ChIP-seq experiment. First of all, the preparation of the protoplasts is very important. It is necessary to control the collapse time so that high-quality protoplasts can be collected. Ultrasound is also very important, the ultrasound time should be controlled, too long or too short will not work. Secondly, the amount of antibody added should be sufficient to facilitate the enrichment of more DNA fragments that bind to the protein. When verifying the quality and quantity of DNA precipitated in the ChIP-seq experiment Qubit Fluorometer was used. Agilent 2100 was used to detect the mass concentration and fragment distribution of DNA, which provides a basis for whether the sample can be used for subsequent library establishment and sequencing experiments.

Overall, this protocol enhances the understanding of the whole genome-wide distribution of epigenetic modifications that regulate pathogenic genes during pathogen infection. This method will contribute to identifying molecular mechanisms of epigenetic modifications and identify new target genes during fungi development and pathogen-induced pathogenesis in M. oryzae and other filamentous fungi.

Materials

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