A chromatin immunoprecipitation optimization method suitable for plant tissues

Abstract

The application discloses a chromatin immunoprecipitation optimization method suitable for plant tissues and belongs to the technical field of plant molecular biology. The whole process of formaldehyde crosslinking, nuclear extraction, chromatin ultrasonic fragmentation, immunological enrichment and DNA purification is optimized, the buffer system is unified, the ultrasonic parameters and the washing scheme are optimized, and the sample loss and non-specific binding are reduced. Meanwhile, the purification mode of phenol-chloroform combined with ethanol precipitation is adopted, and the efficiency and safety are considered. The method has strong operation repeatability, the chromatin fragment distribution is concentrated in 200-600 bp, the DNA yield is high, the method can be stably used for PCR and high-throughput library sequencing, and the problems of uneven fragments, high background noise and low throughput of the traditional plant ChIP method are solved.

Description

Technical Field

[0001] This invention relates to the field of plant molecular biology, specifically to an optimized method for chromatin immunoprecipitation of plant tissues. Background Technology

[0002] Chromatin immunoprecipitation (ChIP) is a core experimental technique for studying protein-DNA interactions in plants, widely used in research fields such as gene expression regulation and epigenetic modification. However, traditional plant tissue ChIP methods have several drawbacks: First, the experimental procedure involves multiple tubes, resulting in significant sample loss during transfer and washing, and poor sample homogeneity. Second, improper chromatin sonication parameters can easily lead to excessively large or fragmented fragments, resulting in uneven fragment distribution and directly affecting subsequent enrichment. Third, non-specific binding is a significant issue, leading to high background noise and resulting in low qPCR enrichment folds and poor signal-to-noise ratios in high-throughput sequencing. Fourth, traditional DNA purification often relies on highly toxic phenol-chloroform, leading to poor experimental safety and difficulty in adapting to high-throughput experimental requirements. Fifth, there are no standardized buffer systems and operating parameters, resulting in poor reproducibility between different researchers and laboratories. Summary of the Invention

[0003] To address the shortcomings of existing technologies, this invention provides an optimized method for chromatin immunoprecipitation of plant tissues, simplifying the operation process, reducing material loss, and improving cross-linking uniformity. It optimizes the sonication strategy to obtain stable and uniform chromatin fragments. Non-specific binding is reduced, significantly lowering background noise. This achieves non-toxic, rapid, and high-throughput DNA purification. A standardized, reproducible, and patentable complete system is established.

[0004] To achieve the above objectives, the present invention provides the following technical solution: an optimized method for chromatin immunoprecipitation of plant tissues includes the following steps: S1 Formaldehyde crosslinking treatment: Take 2-5 g of fresh plant tissue, wash with deionized water, cut into small pieces, place in 30-40 mL of pre-cooled buffer solution No. 1, add 1% formaldehyde (v / v) and mix well; crosslink under vacuum at room temperature for 4-10 min, gently shaking to mix during the process, repeat 2-3 times; add 125 mM glycine to terminate the crosslinking, continue vacuum treatment for 2-5 min; wash 2-4 times with ice-cold sterile water, blot dry, flash freeze in liquid nitrogen, and store at -80℃; S2 Nucleus Extraction: Frozen plant tissue was ground into a fine powder in liquid nitrogen. 2–3 g of powder was added to 30–40 mL of pre-cooled buffer solution No. 1, and the mixture was rotated at 4°C for 30 min. The mixture was then centrifuged at 4000 g at 4°C for 20 min, and the supernatant was discarded. The precipitate was resuspended in buffer solution No. 2 and sonicated. After sonication, the mixture was centrifuged at 10000 rpm at 4°C for 2 min, and the supernatant was collected to obtain the coarse cell nuclei. S3 Chromatin Sonication Fragmentation: Coarse cell nuclei were gently resuspended in 500–800 μL of sonication buffer and incubated on ice for 15–20 min to complete lysis. The sonication output power was set to 10%–20%, with each sonication session lasting 30 s followed by a 30 s pause, for a total cycle time of 25 min. After sonication, the cells were centrifuged at 12000 g at 4℃ for 15 min, and the supernatant was collected to obtain the chromatin solution. A small amount of chromatin sample was mixed with 5% glycerol and analyzed by 2% agarose gel electrophoresis. Fragments concentrated in the 200–600 bp range were considered acceptable. S4 Immunobinding treatment: The chromatin solution was diluted with dilution buffer to a final SDS concentration of less than 0.1%, and 50 μL of Protein A / G magnetic beads were added. The mixture was rotated at 4°C for 1–2 h to remove non-specific binding. The magnetic beads were separated using a magnetic rack, and the supernatant was collected and specific antibody was added at a volume ratio of 5–7 μL / mL. The mixture was incubated at 4°C overnight. A negative control group without antibody was also set up. The washed Protein A / G magnetic beads were then added and the mixture was incubated at 4°C for 2–4 h to capture immune complexes. S5 Specific Chromatin Extraction: Magnetic beads were separated using a magnetic rack. The beads were washed sequentially with low-salt wash buffer, high-salt wash buffer, lithium chloride wash buffer, and TE buffer at 4°C for 10 min each. After each wash, the beads were adsorbed onto the magnetic rack, and the supernatant was removed. Then, 200–250 μL of elution buffer was added, and the mixture was incubated at 65°C for 15 min for elution. This elution was repeated once, and the eluates were combined. 200 mM sodium chloride and 20 μg / mL proteinase K were added to the eluate, and the mixture was shaken at 65°C for 6–12 h to decrosslink. S6 DNA Purification and Recovery: 0.5 M EDTA, 1 M Tris-HCl, and proteinase K solution were added sequentially, mixed, and incubated at 45°C for 2–3 h. RNase A was then added, and the reaction was carried out at 37°C and 350 rpm for 30 min. The supernatant was extracted using a phenol-chloroform system, and anhydrous ethanol, sodium acetate, and glycogen were added. Precipitation was carried out overnight at -20°C. After 75%... After washing with ethanol and drying, the DNA was dissolved in TE buffer and detected by quantitative PCR. A concentration ≥10 ng / μL was sufficient for subsequent PCR or library construction and sequencing.

[0005] Preferably, the first buffer solution consists of: 10 mM Tris-HCl, 10 mM MgCl2, 400 mM sucrose, and pH=8.0.

[0006] Preferably, the second buffer solution consists of: 10 mM Tris-HCl, 10 mM MgCl2, 200 mM sucrose, pH=8.0; the ultrasonic output power in the cell nucleus extraction step is 10%, with a single ultrasonic session of 30 s followed by a 30 s pause, and a total cycle time of 10 min.

[0007] Preferably, the ultrasonic buffer solution consists of: 10 mM Tris-HCl, 10 mM MgCl2, 2 M sucrose, 0.3% SDS (volume fraction), and pH=8.0.

[0008] Preferably, the dilution buffer components are: 10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 (volume fraction), pH=8.0.

[0009] Preferably, the low-salt washing solution comprises: 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 0.1% SDS (volume fraction), 1% Triton X-100 (volume fraction), and pH=8.0.

[0010] Preferably, the high-salt washing solution comprises: 20 mM Tris-HCl, 2 mM EDTA, 450 mM NaCl, 0.1% SDS (volume fraction), 1% Triton X-100 (volume fraction), and pH=8.0.

[0011] Preferably, the lithium chloride washing solution comprises: 10 mM Tris-HCl, 1 mM EDTA, 250 mM lithium chloride, 1% sodium deoxycholate (volume fraction), 1% NP-40 (volume fraction), and pH=8.0.

[0012] Preferably, the TE buffer solution consists of 10 mM Tris-HCl, 1 mM EDTA, and pH=8.0.

[0013] This invention provides an optimized method for chromatin immunoprecipitation in plant tissues. It offers the following advantages: (1) Process optimization and reduced sample loss: The cross-linking and washing operations are integrated to reduce the number of sample transfers and improve the utilization rate and experimental uniformity of plant tissue samples. (2) Standardization of ultrasound parameters: The ultrasound power and cycle time are limited, and the chromatin fragments are stably concentrated in 200-600bp, which greatly improves the fragment uniformity. (3) Effective reduction of background noise: The addition of a magnetic bead pre-cleaning step, combined with a gradient salt solution washing system, significantly reduces non-specific binding and improves the experimental signal-to-noise ratio. (4) System standardization: The formulation and operating parameters of various buffer solutions are unified, and the experimental repeatability between laboratories and personnel is significantly enhanced. (5) Wide applicability: The purification scheme takes into account both routine molecular experiments and high-throughput sequencing, with high DNA yield and a minimum concentration of 10 ng / μL, which meets the requirements of various downstream experiments.

[0014] Figure 1 This is a flowchart of the workflow of the present invention. Detailed Implementation

[0015] The technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings of the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments.

[0016] Examples of the embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and intended to explain the invention, and should not be construed as limiting the invention.

[0017] Please see Figure 1 This invention provides a technical solution: an optimized method for chromatin immunoprecipitation in plant tissues, comprising the following steps: Formaldehyde cross-linking treatment → cell nucleus extraction → chromatin sonication fragmentation → immune binding treatment → specific chromatin extraction → DNA purification and recovery.

[0018] in: Formaldehyde cross-linking treatment: Take 2–5 g of fresh plant tissue, wash with deionized water and cut into small pieces, place in 30–40 mL of pre-cooled buffer solution No. 1, add 1% (v / v) formaldehyde and mix well; cross-link under vacuum at room temperature for 4–10 min, intermittently shaking, repeating 2–3 times; add glycine to a final concentration of 125 mM to terminate cross-linking, continue vacuum treatment for 2–5 min; wash the sample 2–4 times with ice-cold sterile water, blot dry, flash freeze in liquid nitrogen, and store at -80℃. Buffer solution No. 1: 10 mM Tris-HCl, 10 mM MgCl2, 400 mM sucrose, pH=8.0.

[0019] Nucleus extraction: The frozen sample was thoroughly ground into a fine powder in liquid nitrogen, avoiding melting during the grinding process. For every 2–3 g of powder, 30–40 mL of pre-cooled buffer solution No. 1 was added, and the mixture was rotated at 4°C for 30 min. The sample was then centrifuged at 4000 g and 4°C for 20 min, and the supernatant was discarded. The precipitate was resuspended in buffer solution No. 2. The sonication output power was set to 10%, with each sonication cycle lasting 30 s followed by a 30 s pause, for a total cycle time of 10 min. After sonication, the sample was centrifuged at 10000 rpm and 4°C for 2 min, and the supernatant was collected as the crude cell nuclei. Buffer solution No. 2: 10 mM Tris-HCl, 10 mM MgCl2, 200 mM sucrose, pH=8.0.

[0020] Chromatin sonication fragmentation: Take coarse cell nuclei, gently resuspend them in 500–800 μL of sonication buffer, and incubate on ice for 15–20 min to complete lysis. Set the sonication output power to 10%–20%, with a single sonication session of 30 s and a 30 s interval, repeating for 25 min. After sonication, centrifuge at 12000 g, 4℃ for 15 min, and collect the supernatant to obtain the chromatin solution. Mix the sample with 5% glycerol and detect the fragments using 2% agarose gel electrophoresis; fragments within the 200–600 bp range are considered acceptable. Sonication buffer: 10 mM Tris-HCl, 10 mM MgCl2, 2 M sucrose, 0.3% SDS (v / v), pH=8.0.

[0021] Immunobinding treatment: The chromatin solution was diluted with dilution buffer to a SDS volume fraction below 0.1%; 50 μL of Protein A / G magnetic beads were added and rotated at 4°C for 1–2 h to remove non-specific binding; after separating the beads with a magnetic rack, the supernatant was aspirated, and specific antibodies were added at a ratio of 5–7 μL / mL, incubated overnight at 4°C, and a negative control without antibodies was set up; then washed Protein A / G magnetic beads were added, and incubated at 4°C for 2–4 h to capture immune complexes. Dilution buffer: 10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100, pH=8.0.

[0022] Specific chromatin extraction: Magnetic beads were immobilized using a magnetic rack and washed sequentially with low-salt washing buffer, high-salt washing buffer, lithium chloride washing buffer, and TE buffer, rotating for 10 min at 4°C for each wash. After each wash, the magnetic beads were adsorbed and the supernatant was removed. 200–250 μL of elution buffer (100 mM NaHCO3, 1% SDS) was added, and the mixture was incubated at 65°C for 15 min for elution. This process was repeated once, and the elution buffers were combined. 200 mM sodium chloride and 20 μg / mL proteinase K were added to the elution buffer, and the mixture was shaken at 65°C for 6–12 h to decrosslink. Washing buffer formulations: Low-salt wash buffer: 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 0.1% SDS, 1% Triton X-100, pH=8.0; High-salt wash buffer: 20 mM Tris-HCl, 2 mM EDTA, 450 mM NaCl, 0.1% SDS, 1% Triton X-100, pH=8.0; Lithium chloride wash buffer: 10 mM Tris-HCl, 1 mM EDTA, 250 mM lithium chloride, 1% sodium deoxycholate, 1% NP-40, pH=8.0; TE buffer: 10 mM Tris-HCl, 1 mM EDTA, pH=8.0.

[0023] DNA purification and recovery: 0.5 M EDTA, 1 M Tris-HCl, and 10 mg / mL proteinase K were added sequentially, mixed thoroughly after each addition, and briefly centrifuged. The mixture was then incubated at 45°C with shaking for 2–3 h. Next, 10 mg / mL RNase A was added, and the reaction was carried out at 37°C and 350 rpm for 30 min. The supernatant was extracted using a phenol-chloroform system, and 2.5 volumes of anhydrous ethanol, 1 / 10 volume of 3 M sodium acetate, and 2 μL of glycogen were added. The mixture was incubated at -20°C overnight. The precipitate was washed with 75% ethanol, air-dried at room temperature, and the DNA was dissolved in TE buffer. When the DNA concentration was ≥10 ng / μL, it was ready for PCR or library preparation and sequencing, as determined by a quantitative PCR instrument.

[0024] The following are the control experimental data for this scheme: chromatin fragment homogeneity Relatively dispersed (100-600bp) Relatively uniform (200-500bp) DNA yield Typical (10-20 ng / μL) High (80-100 ng / μL) qPCR enrichment factor Generally (1-1.5 times) Higher (2-3 times) Background noise (DNA content in negative control) Typical (2-5 ng / μL) Typical (2-5 ng / μL) Percentage of effective reads from library preparation and sequencing 80-90% 80-90% Experiment time 3 days 2 and a half days Sample loss rate More (generally over 60%) Less (30%) The above description is only a preferred embodiment of the present invention, but the scope of protection of the present invention is not limited thereto. Any equivalent substitutions or modifications made by those skilled in the art within the scope of the technology disclosed in the present invention, based on the technical solution and inventive concept of the present invention, should be covered within the scope of protection of the present invention.

Claims

1. A chromatin immunoprecipitation optimization method suitable for plant tissues, characterized in that, Includes the following steps: S1 Formaldehyde crosslinking treatment: Take 2-5 g of fresh plant tissue, wash with deionized water, cut into small pieces, place in 30-40 mL of pre-cooled buffer solution No. 1, add 1% formaldehyde (v / v) and mix well; crosslink under vacuum at room temperature for 4-10 min, gently shaking to mix during the process, repeat 2-3 times; add 125 mM glycine to terminate the crosslinking, continue vacuum treatment for 2-5 min; wash 2-4 times with ice-cold sterile water, blot dry, flash freeze in liquid nitrogen, and store at -80℃; S2 Nucleus Extraction: Frozen plant tissue was ground into a fine powder in liquid nitrogen. 2–3 g of powder was added to 30–40 mL of pre-cooled buffer solution No. 1, and the mixture was rotated at 4°C for 30 min. The mixture was then centrifuged at 4000 g at 4°C for 20 min, and the supernatant was discarded. The precipitate was resuspended in buffer solution No. 2 and sonicated. After sonication, the mixture was centrifuged at 10000 rpm at 4°C for 2 min, and the supernatant was collected to obtain the coarse cell nuclei. S3 Chromatin Sonication Fragmentation: The coarse cell nuclei were gently resuspended in 500–800 μL of sonication buffer and incubated on ice for 15–20 min to complete lysis. The sonication output power was set to 10%–20%, with each sonication cycle lasting 30 s and a 30 s pause, for a total cycle time of 25 min. After sonication, the cells were centrifuged at 12000 g at 4℃ for 15 min, and the supernatant was collected to obtain the chromatin solution. A small amount of chromatin sample was mixed with 5% glycerol and analyzed by 2% agarose gel electrophoresis. Fragments concentrated in the 200–600 bp range were considered acceptable. S4 Immunobinding treatment: The chromatin solution was diluted with dilution buffer to a final SDS concentration of less than 0.1%, and 50 μL of Protein A / G magnetic beads were added. The mixture was rotated at 4°C for 1–2 h to remove non-specific binding. The magnetic beads were separated using a magnetic rack, and the supernatant was collected and specific antibody was added at a volume ratio of 5–7 μL / mL. The mixture was incubated at 4°C overnight. A negative control group without antibody was also set up. The washed Protein A / G magnetic beads were then added and the mixture was incubated at 4°C for 2–4 h to capture immune complexes. S5 Specific Chromatin Extraction: Magnetic beads were separated using a magnetic rack. The beads were washed sequentially with low-salt wash buffer, high-salt wash buffer, lithium chloride wash buffer, and TE buffer at 4°C for 10 min each. After each wash, the beads were adsorbed onto the magnetic rack, and the supernatant was removed. Then, 200–250 μL of elution buffer was added, and the mixture was incubated at 65°C for 15 min for elution. This elution was repeated once, and the eluates were combined. 200 mM sodium chloride and 20 μg / mL proteinase K were added to the eluate, and the mixture was shaken at 65°C for 6–12 h to decrosslink. S6 DNA Purification and Recovery: 0.5 M EDTA, 1 M Tris-HCl, and proteinase K solution were added sequentially, mixed, and incubated at 45°C for 2–3 h. RNase A was then added, and the reaction was carried out at 37°C and 350 rpm for 30 min. The supernatant was extracted using a phenol-chloroform system, and anhydrous ethanol, sodium acetate, and glycogen were added. Precipitation was carried out overnight at -20°C. After 75%... After washing with ethanol and drying, the DNA was dissolved in TE buffer and detected by quantitative PCR. A concentration ≥10 ng / μL was sufficient for subsequent PCR or library construction and sequencing.

2. The chromatin immunoprecipitation optimization method suitable for plant tissues according to claim 1, characterized in that, The first buffer solution consists of: 10 mM Tris-HCl, 10 mM MgCl2, 400 mM sucrose, and pH=8.

0.

3. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The second buffer solution consists of 10 mM Tris-HCl, 10 mM MgCl2, and 200 mM sucrose, with a pH of 8.

0. In the cell nucleus extraction step, the ultrasonic output power is 10%, with each ultrasonic session lasting 30 seconds followed by a 30-second pause, for a total cycle time of 10 minutes.

4. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The ultrasonic buffer solution consists of: 10 mM Tris-HCl, 10 mM MgCl2, 2 M sucrose, 0.3% SDS (volume fraction), and pH 8.

0.

5. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The dilution buffer components are: 10 mM Tris-HCl, 10 mM EDTA, 150 mM NaCl, 1% Triton X-100 (volume fraction), pH=8.

0.

6. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The low-salt washing solution consists of: 20 mM Tris-HCl, 2 mM EDTA, 150 mM NaCl, 0.1% SDS (volume fraction), 1% Triton X-100 (volume fraction), and pH=8.

0.

7. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The high-salt washing solution consists of: 20 mM Tris-HCl, 2 mM EDTA, 450 mM NaCl, 0.1% SDS (volume fraction), 1% Triton X-100 (volume fraction), and pH=8.

0.

8. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The lithium chloride washing solution consists of: 10 mM Tris-HCl, 1 mM EDTA, 250 mM lithium chloride, 1% sodium deoxycholate (v / v), 1% NP-40 (v / v), and pH=8.

0.

9. The method for optimizing chromatin immunoprecipitation in plant tissues according to claim 1, characterized in that, The TE buffer solution consists of 10 mM Tris-HCl, 1 mM EDTA, and pH 8.0.

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