Liriodendron tulipifera cell nucleus extraction method suitable for CUTTag technology and application of liriodendron tulipifera cell nucleus extraction method

Abstract

The invention discloses a method and a device suitable for CUAMP. The invention discloses a method for extracting liriodendron tulipifera cell nucleuses by a Tag technology and application of the liriodendron tulipifera cell nucleuses. Hybridized liriodendron tulipifera calluses are used as materials, and protoplasts are released through enzymolysis solution vacuumizing and mild enzymolysis for 1.5 h; the method comprises the following steps: sequentially purifying by using a W5 solution and mannitol, detecting the activity by FDA, cracking by using an NE buffer solution, and washing by using a Wash buffer solution in two steps, thereby obtaining 2 * 10 < 6 >-3 * 10 < 6 > high-purity and high-integrity cell nucleuses. The obtained core is clean in background and complete in membrane structure, and can be directly used for CUTamp; tag is used for building a library, so that the magnetic bead capturing efficiency is improved by 46%, and protein-DNA interaction high-throughput sequencing under the condition of low sample size is realized. The method disclosed by the invention is simple and convenient to operate and good in repeatability, and provides key technical support for epigenetic research of rare tree species such as liriodendron tulipifera.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a method for extracting the nuclei of tulip tree cells suitable for CUT&Tag technology and its application. Background Technology

[0002] Chromatin immunoprecipitation (ChIP), a classic technique for elucidating protein-DNA interactions, has been widely used in model plants such as Arabidopsis thaliana and rice. However, ChIP experiments are time-consuming (usually 2-3 days), require large initial sample sizes (leaf or callus ≥2g), have high background signals, and require ultrasonic fragmentation of chromatin, with relatively relaxed requirements on nuclear integrity. For precious and difficult-to-regenerate tree species like Liriodendron chinense, its somatic embryogenesis system is inefficient and its material is scarce, often making it difficult to meet the sample size required for ChIP.

[0003] Cleavage under Targets and Tagmentation is an epigenetic technique used to study protein-DNA interactions. It utilizes antibody-guided transposases to precisely cleave the DNA region where the target protein binds and simultaneously add sequencing adapters, enabling high-resolution, low-background, and low-sample-volume protein-DNA interaction analysis.

[0004] In recent years, Cleavage UnderTargets and Tagmentation (CUT&Tag) technology has gained popularity due to its low sample size (as low as 1×10⁻⁶). 4 The advantages of cellular-level (low background, high resolution) technology have led to its rapid rise in animal and a few model plant studies. The principle involves immobilizing cell nuclei on the surface of magnetic beads, then using a Protein A / G-Tn5 fusion transposase to cleave and ligate sequencing adapters in situ under antibody targeting, directly obtaining an amplifiable library. This skips the sonication and end-repair steps of traditional ChIP, significantly shortening the experimental cycle. However, CUT and Tag technologies have extremely high requirements for the purity and integrity of the cell nucleus and the integrity of the membrane surface glycoprotein structure: any residual cell debris, organelles, or enzymatic digestion byproducts will compete for magnetic bead binding sites, leading to non-specific cleavage by the Tn5 enzyme, resulting in high background or library failure.

[0005] Currently, density gradient centrifugation (such as Percoll or sucrose gradient) is commonly used in the plant field to extract cell nuclei. However, this technique has revealed the following problems in Liriodendron tulipifera: High levels of impurities: Liriodendron tulipifera callus tissue is rich in phenols, polysaccharides, and lignin precursors, which cannot be effectively removed by gradient centrifugation. Furthermore, cell nuclei extracted by density gradient centrifugation often clump together, resulting in a magnetic bead capture efficiency of less than 10%, severely impacting subsequent CUT & Tag experiments; Low yield: Traditional methods yield only (3-5) × 10⁻⁶ cells per gram of callus tissue. 4 The number of cell nuclei is far lower than the (1-2)×10⁻⁶ recommended for CUT&Tag library construction. 5 One initial quantity;

[0006] Therefore, there is currently no feasible method for extracting cell nuclei from *Liriodendron tulipifera* that can support the stable implementation of CUT and Tag technologies. This directly restricts the analysis of its epigenetic regulatory network (such as histone modifications and transcription factor binding sites), becoming a key technical bottleneck hindering molecular breeding and the discovery of superior gene resources in *Liriodendron tulipifera*. Developing an efficient method for extracting *Liriodendron tulipifera* cell nuclei has become an urgent need to break through the application of CUT & Tag technologies in this species. Summary of the Invention

[0007] The purpose of this invention is to provide an efficient method for extracting the nuclei of tulip tree cells suitable for CUT&Tag technology and its application.

[0008] To achieve the above objectives, the present invention provides the following technical solution: In a first aspect, this application provides a method for extracting the nuclei of Liriodendron tulipifera cells suitable for CUT&Tag technology.

[0009] Secondly, this application provides a kit for extracting nuclear cells from *Liriodendron chinense* cells, comprising an enzymatic digest, W5 solution, NE buffer, Wash buffer, and an instruction manual.

[0010] Thirdly, the application of the *Liriodendron tulipifera* cell nuclei obtained by the method provided in this application in CUT&Tag technology.

[0011] The first aspect of this application provides a method for extracting the nuclei of tulip tree cells suitable for CUT&Tag technology, comprising the following steps:

[0012] (1) Sample pretreatment: Select callus tissue of hybrid tulip tree in good growth condition, separate the callus tissue from the somatic embryo under sterile conditions, remove the somatic embryo tissue, and obtain relatively pure callus material, which is the pretreated sample.

[0013] (2) Enzymatic hydrolysis of cell walls: The pretreated sample was added to the enzymatic hydrolysate, and under dark conditions, a vacuum was first drawn, and then enzymatic hydrolysis was carried out on a horizontal shaker to obtain protoplasts;

[0014] (3) Check the protoplast status: Take the enzyme hydrolysate for microscopic observation to assess the protoplast release;

[0015] (4) Protoplast purification: First wash the protoplasts with W5 solution, then resuspend them with 0.6M mannitol and centrifuge to complete the purification of the protoplasts;

[0016] (5) Protoplast viability test: Mix protoplasts with an equal volume of fluorescein diacetate FDA working solution, incubate in the dark, and then observe their viability status through a fluorescence microscope.

[0017] (6) Cell nucleus purification: Protoplasts were lysed with NE buffer and then washed with Wash buffer to obtain pure cell nuclei.

[0018] Further, in step (2), the pretreated sample is added to the enzymatic hydrolysate, and under dark conditions at 27°C, it is first vacuumed for 30 minutes, and then enzymatically hydrolyzed on a horizontal shaker at 40 rpm for 1.5 hours to obtain protoplasts;

[0019] The enzymatic hydrolysate consisted of the following components: 1.5% Cellucase cellulase R-10, 0.5% Macerozyme, 0.1% Pectolyase Y-23, 0.6M mannitol, 80mM KCl, 20mM MES, and 10mM CaCl2; the volume-to-weight ratio of the enzymatic hydrolysate to the pretreated sample was 10mL:0.7g.

[0020] Further, in step (4), an equal volume of pre-cooled W5 solution is added to the enzymatic hydrolysate, mixed, and filtered through a 40 μm cell sieve. The filtrate is centrifuged and resuspended in W5 solution and 0.6 M mannitol to obtain a purified protoplast suspension. The W5 solution consists of 154 mM NaCl, 125 mM CaCl2·2H2O, 5 mM KCl, and 2 mM MES (pH 5.7).

[0021] Furthermore, in step (5), the FDA working solution is prepared by diluting the FDA stock solution 100 times with 0.6M mannitol, wherein the stock solution is 5 mg FDA dissolved in 5 mL acetone and stored at -20°C protected from light.

[0022] Further, in step (6), the composition of the NE buffer is: 1M HEPES-KOH (pH 7.9), 1M KCl, 2Mspermidine, 10% Triton-X100, 20% glycerol, and 1 Roche Complete Protease Inhibitor EDTA-Free tablet.

[0023] Furthermore, the Wash buffer consists of: 1M HEPES-KOH (pH 7.5), 5M NaCl, 2Mspermidine, and 1 Roche Complete Protease Inhibitor EDTA-Free tablet.

[0024] Furthermore, in step (6), the centrifugation conditions are: 23°C, 150×g, 2 minutes, with acceleration / deceleration rates of 2–3.

[0025] Furthermore, in step (6), the number of cell nuclei extracted by the method can reach 2 × 10⁻⁶. 6 -3×10 6 One, suitable for subsequent CUT&Tag experiments.

[0026] A second aspect of this application provides a kit for extracting nuclear cells from *Liriodendron chinense* cells, comprising an enzymatic digest, W5 solution, NE buffer, Wash buffer, and instructions for use.

[0027] The third aspect of this application provides a method for the application of tulip tree cell nuclei obtained in CUT&Tag, using the cell nuclei as starting material, binding them with ConA magnetic beads, and then incubating them with an antibody-transposase to construct a protein-DNA interaction library.

[0028] Beneficial effects: This invention has high extraction efficiency, significantly better cell nucleus purity and integrity than traditional methods, extremely low sample requirements, is suitable for rare materials, greatly improves the success rate of CUT&Tag library construction, has strong technical stability and reproducibility, is easy to operate, and does not require expensive equipment, providing key technical support for epigenetic research on Liriodendron tulipifera.

[0029] Compared with the prior art, the present invention has the following advantages:

[0030] (1) This invention significantly shortens the cell wall degradation time of tulip callus (completed in 1.5 hours) by optimizing the enzymatic hydrolysate formulation (containing Cellucase, Macerozyme, and Pectolyase) and using a combination of vacuuming and gentle shaking, and increases the protoplast release efficiency by more than 30%, avoiding the lengthy process of traditional methods (such as density gradient centrifugation).

[0031] (2) The present invention adopts the "protoplast lysis-two-step buffer purification" strategy (NE buffer lysis + Wash buffer washing) to remove cell debris and organelle impurities. DAPI staining shows that the background cleanliness is more than 95% and the cell nucleus integrity rate is >90%, which fully meets the stringent requirements of CUT and Tag technology for "no impurities and no debris".

[0032] (3) This invention requires only 0.7g of callus tissue (traditional ChIP requires ≥2g) to obtain 2×10 6 -3×10 6 High-purity cell nuclei are particularly suitable for precious tree species such as tulip trees or non-renewable somatic embryo materials, solving the bottleneck of not being able to conduct epigenetic research on rare samples.

[0033] (4) The cell nuclei extracted by this invention were tested by magnetic bead capture efficiency. The protoplast lysis-two-step buffer purification method improved the cell nuclei capture efficiency by 46% compared with the traditional density gradient method (Table 1), which improved the sequencing success rate. The main peak of the library insertion fragment was distributed in 200-500bp, which fully met the Illumina sequencing standard and achieved a breakthrough of "low sample size-high data quality".

[0034] (5) All reagents in the optimized scheme of this invention (such as W5 solution and NE buffer) have been verified by strict component ratio and storage conditions (such as storage at 4°C for 1-3 months), and the experimental coefficient of variation (CV) between batches is <5%, ensuring that different laboratories or different operators can obtain consistent results.

[0035] (6) The present invention only requires a regular centrifuge, a fluorescence microscope and conventional molecular reagents throughout the process, avoiding dependence on ultra-high speed centrifuges, flow cytometers, etc., reducing the technical threshold and making it suitable for promotion in ordinary plant molecular laboratories.

[0036] (7) This invention is the first to achieve CUT & Tag detection of histone modifications (such as H3K27me3) and transcription factors (such as LcbZIP9) at the nuclear level of Liriodendron tulipifera cells, filling the technical gap in the study of the epigenetic regulatory mechanism of this species and providing new targets for molecular breeding. Attached Figure Description

[0037] To more clearly illustrate the technical solutions in the embodiments of this application, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0038] Figure 1 This is a microscopic observation of the callus tissue of Liriodendron tulipifera after enzymatic hydrolysis for 1.5 hours, according to the present invention.

[0039] Figure 2 This invention presents a fluorescence observation diagram of protoplast viability detection using fluorescein diacetate (FDA).

[0040] Figure 3 This invention uses DAPI to detect the fluorescence observation pattern of cell nucleus purity and integrity.

[0041] Figure 4 Electrophoresis diagram showing the distribution of successfully constructed text fragments in this invention.

[0042] Figure 5 The peak diagram shows the successful database construction of CUT&Tag in this invention. Detailed Implementation

[0043] The present invention will now be described in further detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the scope of protection of the present invention.

[0044] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0045] In this application, the term "and / or" describes the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, or B existing alone. A and B can be singular or plural. The character " / " generally indicates that the preceding and following related objects have an "or" relationship.

[0046] In this application, "-one less" means one or more, and "more than" means two or more. "-one less item (item) below" or similar expressions refer to any combination of these items, including any combination of single or multiple items. For example, "-one less item (item) in a, b, or c", or "-one less item (item) in a, b, and c", can all mean: a, b, c, ab (i.e., a and b), ac, bc, or abc, where a, b, and c can be single or multiple.

[0047] It should be understood that in the various embodiments of this application, the order of the above processes does not imply the order of execution. Some or all steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of this application.

[0048] This application provides a method for extracting the nuclei of Liriodendron tulipifera cells suitable for CUT and Tag technologies, comprising the following steps:

[0049] (1) Sample pretreatment: Select callus tissue of hybrid tulip tree in good growth condition, separate the callus tissue from the somatic embryo under sterile conditions, remove the somatic embryo tissue, and obtain relatively pure callus material, which is the pretreated sample.

[0050] (2) Enzymatic hydrolysis of cell walls: The pretreated sample was added to the enzymatic hydrolysate, and under dark conditions, a vacuum was first drawn, and then enzymatic hydrolysis was carried out on a horizontal shaker to obtain protoplasts;

[0051] (3) Check the protoplast status: Take the enzyme hydrolysate for microscopic observation to assess the protoplast release;

[0052] (4) Protoplast purification: First wash the protoplasts with W5 solution, then resuspend them with 0.6M mannitol and centrifuge to complete the purification of the protoplasts;

[0053] (5) Protoplast viability test: Mix protoplasts with an equal volume of fluorescein diacetate FDA working solution, incubate in the dark, and then observe their viability status through a fluorescence microscope.

[0054] (6) Cell nucleus purification: Protoplasts were lysed with NE buffer and then washed with Wash buffer to obtain pure cell nuclei.

[0055] In some embodiments, in step (2), the pretreated sample is added to the enzymatic hydrolysate, and under dark conditions at 27°C, it is first evacuated for 30 minutes, and then incubated on a horizontal shaker at 40 rpm for 1.5 hours to obtain protoplasts.

[0056] In some embodiments, the enzymatic hydrolysate consists of the following components: 1.5% Cellucase cellulase R-10, 0.5% Macerozyme, 0.1% Pectolyase Y-23, 0.6M mannitol, 80mM KCl, 20mM MES, and 10mM CaCl2; the volume-to-weight ratio of the enzymatic hydrolysate to the pretreated sample is 10mL:0.7g.

[0057] Further, in step (4), an equal volume of pre-cooled W5 solution is added to the enzymatic hydrolysate, mixed, and filtered through a 40 μm cell sieve. The filtrate is centrifuged and resuspended in W5 solution and 0.6 M mannitol to obtain a purified protoplast suspension. The W5 solution consists of 154 mM NaCl, 125 mM CaCl2·2H2O, 5 mM KCl, and 2 mM MES (pH 5.7).

[0058] Furthermore, in step (5), the FDA working solution is prepared by diluting the FDA stock solution 100 times with 0.6M mannitol, wherein the stock solution is 5 mg FDA dissolved in 5 mL acetone and stored at -20°C protected from light.

[0059] In some embodiments, in step (6), the composition of the NE buffer is: 1M HEPES-KOH (pH 7.9), 1M MKCl, 2M spermidine, 10% Triton-X100, 20% glycerol, and 1 Roche Complete Protease Inhibitor EDTA-Free tablet.

[0060] In some embodiments, the Wash buffer consists of: 1M HEPES-KOH (pH 7.5), 5M NaCl, 2Mspermidine, and 1 Roche Complete Protease Inhibitor EDTA-Free tablet.

[0061] In some embodiments, in step (6), the centrifugation conditions are: 23°C, 150×g, 2 minutes, and the acceleration / deceleration rate is 2–3.

[0062] In some embodiments, in step (6), the number of cell nuclei extracted by the method can reach 2 × 10⁻⁶. 6 -3×10 6 One, suitable for subsequent CUT&Tag experiments.

[0063] A second aspect of this application provides a kit for extracting nuclear cells from *Liriodendron chinense* (tulip tree) cells, comprising an enzymatic digest, W5 solution, NE buffer, Wash buffer, and an instruction manual.

[0064] The third aspect of this application provides the application of a method for obtaining tulip tree cell nuclei in CUT&Tag. Using the cell nuclei as starting material, the nuclei are bound to ConA magnetic beads and then incubated with an antibody-transposase to construct a protein-DNA interaction library.

[0065] Example 1

[0066] The present invention provides a method for extracting the nuclei of *Liriodendron chinense* cells suitable for CUT&Tag technology, comprising the following steps:

[0067] (1) Sample pretreatment: Select callus tissue of hybrid tulip tree in good growth condition, separate the callus tissue from the somatic embryo under sterile conditions, remove the somatic embryo tissue, and obtain relatively pure callus material, which is the pretreated sample.

[0068] (2) Enzymatic hydrolysis of cell walls: The pretreated sample was added to the enzymatic hydrolysate, which consisted of the following components: 1.5% Cellucase cellulase R-10, 0.5% Macerozyme, 0.1% Pectolyase Y-23, 0.6M mannitol, 80mM KCl, 20mM MES (pH 5.7), and 10mM CaCl2. Under dark conditions at 27°C, the sample was first vacuumed for 30 minutes, and then incubated on a horizontal shaker at 40 rpm for 1.5 hours to obtain protoplasts.

[0069] (3) Check the protoplast status: Take the enzymatic hydrolysate for microscopic observation to assess the protoplast release. If there are a large number of cell clumps, the enzymatic hydrolysis time can be extended appropriately, but the total time should not exceed 2 hours. The volume-to-weight ratio of the enzymatic hydrolysate to the pretreated sample is 10 mL: 0.7 g.

[0070] (4) Protoplast purification: Add an equal volume of pre-cooled W5 solution to the enzymatic hydrolysate, mix well, filter through a 40μm cell sieve, centrifuge the filtrate, and resuspend it in W5 solution and 0.6M mannitol to obtain a purified protoplast suspension; the composition of W5 solution is: 154mM NaCl, 125mM CaCl2·2H2O, 5mM KCl, 2mM MES (pH 5.7).

[0071] (5) Protoplast viability test: Mix protoplasts with an equal volume of fluorescein diacetate FDA working solution, incubate in the dark, and observe their viability under a fluorescence microscope; FDA working solution is prepared by diluting FDA stock solution 100 times with 0.6M mannitol, the stock solution is 5mg FDA dissolved in 5mL acetone, and stored at -20℃ in the dark.

[0072] (6) Cell nucleus purification: After centrifuging and collecting protoplasts, resuspend them in pre-cooled NE buffer, incubate on ice, centrifuge again, discard the supernatant, and resuspend the cell nuclei in Wash buffer. Repeat washing as needed to obtain a pure cell nucleus suspension. The composition of NE buffer is: 1M HEPES-KOH (pH 7.9), 1M KCl, 2M spermidine, 10% Triton-X100, 20% glycerol, and 1 Roche Complete Protease Inhibitor EDTA-Free tablet. The composition of Wash buffer is: 1M HEPES-KOH (pH 7.5), 5M NaCl, 2M spermidine, and 1 Roche Complete Protease Inhibitor EDTA-Free tablet. Centrifugation conditions are: 23℃, 150×g, 2 minutes, with acceleration / deceleration rates of 2–3. The number of extracted cell nuclei can reach 2×10⁻⁶. 6-3×10 6 One, suitable for subsequent CUT&Tag experiments.

[0073] Example 2

[0074] This invention provides a kit for extracting nuclear cells from *Liriodendron chinense* (tulip tree) cells, comprising an enzymatic digest, W5 solution, NE buffer, Wash buffer, and an instruction manual.

[0075] Example 3

[0076] CUT&Tag Library Construction and Application

[0077] 1. Initial amount of cell nucleus

[0078] Take 2 × 10⁶ Liriodendron tulipifera cell nuclei obtained in Example 1 6 -3×10 6 Each sample was resuspended in 100 μL of NE buffer and counted with trypan blue to show a survival rate of >90%.

[0079] 2. ConA magnetic bead bonding

[0080] Add 10 μL of activated ConA-magnetic beads (Active Motif, #53132), and gently shake at room temperature for 20 min; discard the supernatant using a magnetic rack, and resuspend in 50 μL of pre-cooled Antibody Buffer.

[0081] 3. Antibody-transposase incubation

[0082] Primary antibody: CST H3K27me3(#9733) 1:100, overnight at 4℃;

[0083] Secondary antibody: Rabbit anti-IgG-A / G-Tn5 (Active Motif, #53138) 1:100, incubated at room temperature for 60 min; wash 3 times with 200 μL of Dig-wash buffer each time.

[0084] 4. Fragmentation and Termination

[0085] Add 50 μL Tn5 reaction buffer (containing 0.04 μM pA / G-Tn5), incubate at 37 °C for 60 min; then add 2 μL 10% SDS + 0.5 pg DNA spike-in, incubate at 55 °C for 20 min to terminate.

[0086] 5. Library amplification

[0087] Transfer the supernatant to a PCR tube, add 50 μL of pretreated DNA ExtractBeads Pro, mix well, and incubate at room temperature for 20 min; discard the supernatant using a magnetic rack, and wash twice with 1×B&W Buffer; when the surface of the magnetic beads is no longer reflective, add 15 μL of ddH2O; add 25 μL of 2×CAM, 5 μL each of N5xx / N7xx, and incubate for 15 cycles (72℃ for 1 min).

[0088] VAHTS DNA Clean Beads purification, elution with 22 μL EB, detection with Agilent 2100: main peak 200-500 bp, concentration ≥2 nM, Q30 ≥90%.

[0089] 6. Sequencing and Data Analysis

[0090] Illumina NovaSeq 6000PE150;

[0091] Bowtie2 compared to the Liriodendron tulipifera genome, MACS2 peak calling, ≥2fold vs IgG control, FDR < 0.01.

[0092] Results: The number of H3K27me3 peaks was 12,847, with 78% overlap with gene promoters, confirming that the cell nucleus is suitable for epigenetic regulation research.

[0093] The application of tulip tree cell nuclei obtained by a method of the present invention in CUT and Tag involves using the cell nuclei as starting material, binding them with ConA magnetic beads, and then incubating them with an antibody-transposase to construct a protein-DNA interaction library.

[0094] Example 4

[0095] A method for constructing a Liriodendron tulipifera library suitable for CUT&Tag according to the present invention includes the following steps: (1) incubating cell nuclei with ConABeads:

[0096] Transfer 100 μL of cell nuclei into an eight-tube containing activated ConABeads, invert to mix, and incubate at room temperature for 20 min, inverting to mix 2-3 times during the incubation period; centrifuge briefly, place on a magnetic rack, and discard the supernatant after the solution has clarified.

[0097] (2) Primary antibody incubation:

[0098] Add 50 μL of pre-chilled Antibody Buffer to resuspend the cell nucleus-magnetic bead complex in each sample; add 1 μL of antibody: CST H3K27me3 Rabbit mAb, invert and mix well; briefly centrifuge to collect the liquid at the bottom of the tube, and place the eight-tube strip at 4°C overnight (negative control: no primary antibody added).

[0099] (3) Secondary antibody incubation:

[0100] Dilute the secondary antibody 1:100 with Dig-wash Buffer, 50 μL per sample; take the eight-tube strip from step 2, briefly centrifuge to collect the reaction solution, place the eight-tube strip on a magnetic rack, and discard the supernatant after the solution becomes clear; add the diluted secondary antibody, gently invert several times to mix the antibody with the nuclear-magnetic bead complex, and incubate at room temperature for 60 min; briefly centrifuge, place on a magnetic rack, and discard the supernatant; add 200 μL of Dig-wash Buffer, invert several times to ensure the buffer is fully mixed with the nuclear-magnetic bead complex, and repeat the washing process twice (3 times in total).

[0101] (4) pA / G-Tn5 incubation:

[0102] Add 2 μL of pA / G-Tn5 to 98 μL of Dig-300 Buffer and mix to a final concentration of 0.04 μM. Apply 100 μL to each sample. Take the eight-tube strip from step 3, centrifuge briefly, and discard the supernatant. Add the diluted pA / G-Tn5 transposon and invert several times to ensure the transposon is thoroughly mixed with the nuclear-magnetic bead complex. Incubate at room temperature for 1 hour. Centrifuge briefly and discard the supernatant. Add 200 μL of Dig-300 Buffer and invert several times to ensure the buffer is thoroughly mixed with the nuclear-magnetic bead complex. Repeat the washing process twice (for a total of 3 times).

[0103] (5) Fragmentation:

[0104] Take 40 μL of Dig-300 Buffer, add 10 μL of 5x TTBL, and mix well; take the eight-tube strip from step 4, briefly centrifuge, place on a magnetic rack, and discard the supernatant; add 50 μL of diluted TTBL to each sample and mix well; place the eight-tube strip in a PCR instrument and incubate at 37°C for 60 min (keep the PCR instrument open); briefly centrifuge, add 2 μL of 10% SDS and 0.5 pg DNASpike-in, invert to mix, and incubate at 55°C for 20 min, inverting 2-3 times during incubation; briefly centrifuge, place the eight-tube strip on a magnetic rack, let stand for about 2-3 min, carefully transfer the supernatant to a new eight-tube strip, and discard the magnetic beads.

[0105] (6) DNAExtractBeads Pro treatment:

[0106] Resuspend DNA Extract Beads Pro thoroughly using a pipette. Transfer 25 μL of DNA Extract Beads Pro to a 1.5 mL EP tube, activate DNA Extract Beads Pro with 200 μL 1x B&W Buffer, and finally resuspend DNA Extract Beads Pro with 50 μL 2x B&W Buffer.

[0107] (7) DNA extraction:

[0108] Add 50 μL of DNAExtractBeads Pro from step 6 to the supernatant transferred in step 5, mix thoroughly, and incubate at room temperature for 20 min, inverting and mixing 2-3 times during incubation; centrifuge briefly, place the eight-tube strip on a magnetic rack, let stand for about 2-3 min, and carefully remove the supernatant; keep the PCR tube on the magnetic rack, add 200 μL of 1x B&W Buffer, incubate at room temperature for 30 s, carefully remove the supernatant, and repeat once; open the cap and let stand at room temperature for 5 min until there is no liquid residue in the tube and the surface of the magnetic beads is no longer reflective; add 15 μL of ddH2O to resuspend the DNAExtractBeads Pro.

[0109] (8) Library expansion:

[0110] The following components were prepared in sterile PCR tubes as shown in Table 1:

[0111] Table 1

[0112]

[0113] Gently mix using a pipette, and proceed with the following reaction procedure in the PCR instrument as shown in Table 2:

[0114] Table 2

[0115]

[0116] (9) Glue running verification:

[0117] Place the eight-tube strip from step 8 on a magnetic rack, take 4 μL of PCR product, add 1 μL of loading buffer, mix well by pipetting, and then run a gel to verify whether a ladder-like band of 200 bp-1 kbp is amplified. The results obtained from validating the established library in Example 2 are as follows... Figure 4 As shown. Figure 4 As shown, the library preparation product exhibits diffuse bands, mainly concentrated in the approximately 200-300bp range, which meets the requirements for sequencing. Furthermore, no primer dimers or adapter residues were detected, indicating that the method of this invention can stably obtain high-quality sequencing libraries.

[0118] (10) Purification of PCR products:

[0119] Vortex the VAHTSDNA Clean Beads to mix thoroughly, and add 100 μl to the PCR product. Vortex or pipette 10 times to ensure homogeneity. Incubate at room temperature for 5 min. Briefly centrifuge the reaction tube and place it on a magnetic rack to separate the beads and liquid. Once the solution is clear, carefully remove the supernatant, being careful not to disturb the beads. Keep the PCR tube on the magnetic rack and add 200 μl of freshly prepared 80% ethanol to rinse the beads. Incubate at room temperature for 30 sec, carefully remove the supernatant, and repeat once, for a total of two rinses. Keep the PCR tube on the magnetic rack and air dry for 3-5 min. After the beads are dry, remove the PCR tube from the magnetic rack and add 22 μl of... Elute twice with ddH2O, vortex or pipette 10 times to mix the magnetic beads thoroughly, and incubate at room temperature for 10 min. Briefly centrifuge the PCR tube, place it on a magnetic rack to separate the magnetic beads and liquid, and after the solution is clear, carefully aspirate 20 μl of supernatant and transfer it to a new EP tube, and store at -30 to -15℃.

[0120] (11) Document quality inspection:

[0121] Once the library shows a peak at 200-300bp, sequencing can be scheduled. The peak profile of the CUT&Tag library successfully established in Example 2 is shown below. Figure 5 As shown. Figure 5 As shown, the library fragments are mainly concentrated in the 200-300bp range, with sharp peaks and low background signal, indicating that the method of this invention can stably obtain sequencing libraries with appropriate fragment lengths and high purity, meeting the conditions for sequencing.

[0122] Example 5

[0123] Biomaterials:

[0124] The genotypes of hybrid tulip trees and the callus materials at relevant stages used in the following examples were provided by the laboratory.

[0125] Main reagents:

[0126] The domestically produced reagents D-Mannitol, MES, NaCl, KCl, CaCl2, glycerol, 40μm cell filter, and cellulose microporous filter used in the following examples were all purchased from Sangon Biotech (Shanghai) Co., Ltd.; HEPES-KOH was purchased from Shanghai Beyotime Biotechnology Co., Ltd.; and spermidine was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. The imported reagents cellulase R-10 and analyte R-10 used in the following examples were purchased from Yakult, pectinase Y-23 was purchased from Japan, and Protease Inhibitor EDTA-Free was purchased from MCE.

[0127] Pre-treated samples

[0128] Selected callus tissues of hybrid tulip trees in good growth condition, placed in a clean bench, and separated from the somatic embryos under sterile conditions using a syringe. The somatic embryo tissues were removed as much as possible to obtain relatively pure callus material, which is the pretreated sample.

[0129] Enzymatic hydrolysis of cell walls

[0130] The enzyme digest was prepared according to the preset concentration and ratio and incubated in a 55°C water bath for 10 minutes. Subsequently, the enzyme digest was filtered using a 0.22μm cellulose microporous membrane filter and collected in a sterile Erlenmeyer flask. The pretreated sample was added to the Erlenmeyer flask and gently shaken to ensure the enzyme digest fully submerged the sample. The Erlenmeyer flask was sealed with aluminum foil and vacuumed for 30 minutes at 27°C in the dark. The flask was then sealed and incubated on a horizontal shaker at 40 rpm for 1.5 hours. In this example, the protoplasts were observed under a microscope after 1.5 hours of enzymatic digestion of *Liriodendron tulipifera* callus. Figure 1 As shown. Figure 1 As shown, after repeated enzymatic digestion, there were no large clumps in the tissue cells, and the cell clusters were relatively dispersed.

[0131] Check protoplast status

[0132] After enzymatic hydrolysis, take 10 μL of the reaction solution and place it on a glass slide for microscopic observation to assess the release of protoplasts. If a large number of cell clumps are observed, the hydrolysis time can be appropriately extended to improve the hydrolysis effect, but the total hydrolysis time should not exceed 2 hours to avoid adversely affecting cell viability.

[0133] Protoplast purification

[0134] After passing the test, add an equal volume of pre-chilled W5 solution (pre-chilled on ice) to the enzymatic reaction solution and gently mix. Filter the protoplast mixture through a 40 μm cell sieve. Centrifuge the filtered mixture at 23°C, acceleration / deceleration level 2–3, and 10 × g for 2 minutes, then remove the supernatant. Add twice the volume of ice-chilled W5 solution to the pellet, repeat the centrifugation process under the same conditions, and gently mix to fully suspend the protoplasts. Then add an equal volume of 0.6 M mannitol solution to the cell pellet, and repeat the centrifugation and resuspension steps once. A stable protoplast suspension is finally obtained. The suspension can be briefly stored on ice for subsequent experiments.

[0135] Protoplast Viability Detection

[0136] Protoplast viability was assessed using fluorescein diacetate (FDA). FDA working solution and protoplast suspension were mixed at a 1:1 volume ratio, incubated in the dark for 5 minutes, and then observed under a fluorescence microscope. The viability and cell number of the protoplasts were evaluated based on the fluorescence signal. In this example, the viability of the extracted protoplasts was detected as follows: Figure 2 As shown. Figure 2 As shown, the extracted protoplasts have intact morphology, clear boundaries, and strong fluorescence signals, indicating that they have high viability and integrity.

[0137] Cell nuclear purification

[0138] Take the required number of protoplasts and transfer them to 1.5 mL centrifuge tubes. Centrifuge at 700 × g for 5 minutes at room temperature and discard the supernatant. Add 100 μL of pre-chilled NE buffer to each sample and gently pipette to resuspend the cells. Incubate on ice for 10 minutes. Then centrifuge again at 3000 rpm for 5 minutes at room temperature and discard the supernatant. Add 100 μL of Wash buffer to each sample to resuspend the cell nuclei and briefly incubate at room temperature. If subsequent microscopic observation reveals excessive cell nuclei or an unclean background, further washing can be performed: add 500 μL of Wash buffer to the sample, centrifuge at 700 × g for 5 minutes at room temperature, discard the supernatant, and finally resuspend the cell nuclei in 100 μL of Wash buffer for subsequent analysis.

[0139] Nuclear fluorescence detection

[0140] The obtained resuspension of cell nuclei was mixed with DAPI dye at a 1:1 volume ratio, incubated in the dark for 5 minutes, and then observed under a fluorescence microscope. The integrity, number, and background impurities of the cell nuclei were assessed using fluorescence imaging. The counting results showed that the number of extracted cell nuclei reached 2 × 10⁻⁶. 6 -3×10 6The number of nuclei obtained met the requirements for subsequent CUT&Tag experiments, indicating that the obtained cell nuclei possessed good integrity and purity. In this embodiment, the extracted cell nuclei were counted and examined under a microscope as follows: Figure 3 As shown. Figure 3 As shown, the cell nuclei extracted by the method of this invention appear as dispersed single nuclei after staining, with no obvious aggregation. The cell nuclei have bright fluorescence signals and round morphology, indicating that they have intact structures and high purity, making them suitable for subsequent CUT&Tag experiments.

[0141] By comparing the nucleus capture capabilities of traditional density gradient centrifugation and the protoplast lysis-two-step buffer purification method used in this example, the number of nuclei in the nucleus extract before and after magnetic bead capture was compared, and the nucleus capture rates of the two methods were calculated. The statistical results are shown in Table 3. As can be seen from the table, the nuclei extracted using the protoplast lysis-two-step buffer purification method of this invention have a higher absolute number of nuclei and a higher capture rate, laying the foundation for the success of subsequent experiments.

[0142] Table 3. Number of cell nuclei in the cell nucleus extract before and after magnetic bead capture.

[0143]

[0144] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope. The scope of protection of the present invention is defined by the appended claims, specification, and their equivalents.

Claims

1. A method for extracting the nuclei of Liriodendron tulipifera cells suitable for CUT&Tag technology, characterized in that... Includes the following steps: (1) Sample pretreatment: Select callus tissue of hybrid tulip tree in good growth condition, separate the callus tissue from the somatic embryo under sterile conditions, remove the somatic embryo tissue, and obtain relatively pure callus material, which is the pretreated sample. (2) Enzymatic hydrolysis of cell walls: The pretreated sample was added to the enzymatic hydrolysate, and under dark conditions, a vacuum was first drawn, and then enzymatic hydrolysis was carried out on a horizontal shaker to obtain protoplasts; (3) Check the protoplast status: Take the enzyme hydrolysate for microscopic observation to assess the protoplast release; (4) Protoplast purification: First wash the protoplasts with W5 solution, then resuspend them with 0.6M mannitol and centrifuge to complete the purification of the protoplasts; (5) Protoplast viability test: Mix protoplasts with an equal volume of fluorescein diacetate FDA working solution, incubate in the dark, and then observe their viability status through a fluorescence microscope. (6) Cell nucleus purification: Protoplasts were lysed with NE buffer and then washed with Wash buffer to obtain pure cell nuclei.

2. The extraction method according to claim 1, characterized in that: In step (2), the pretreated sample is added to the enzymatic hydrolysate. Under dark conditions at 27°C, the sample is first vacuumed for 30 minutes, and then enzymatically hydrolyzed at 40 rpm for 1.5 hours on a horizontal shaker to obtain protoplasts. The enzymatic hydrolysate consists of the following components: 1.5% Cellucase cellulase R-10, 0.5% Macerozyme, 0.1% Pectolyase Y-23, 0.6M mannitol, 80mM KCl, 20mM MES, and 10mM CaCl2; the volume-to-weight ratio of the enzymatic hydrolysate to the pretreated sample is 10mL:0.7g.

3. The extraction method according to claim 1, characterized in that: In step (4), an equal volume of pre-cooled W5 solution is added to the enzymatic hydrolysate, mixed, and filtered through a 40 μm cell sieve. The filtrate is centrifuged and resuspended in W5 solution and 0.6 M mannitol to obtain a purified protoplast suspension. The W5 solution consists of 154 mM NaCl, 125 mM CaCl2·2H2O, 5 mM KCl, and 2 mM MME.

4. The extraction method according to claim 1, characterized in that: In step (5), the FDA working solution is prepared by diluting the FDA stock solution 100 times with 0.6M mannitol. The stock solution is 5 mg FDA dissolved in 5 mL acetone and stored at -20°C in the dark.

5. The extraction method according to claim 1, characterized in that: In step (6), after centrifuging and collecting the protoplasts, they are resuspended in pre-cooled NE buffer, incubated on ice, centrifuged again, the supernatant is discarded, and the cell nuclei are resuspended in Wash buffer. Washing is repeated if necessary to obtain pure cell nuclei. The composition of the NE buffer is: 1M pH 7.9 HEPES-KOH, 1M KCl, 2M spermidine, 10% Triton-X100, 20% glycerol, and 1 tablet of Roche Complete Protease Inhibitor EDTA-Free.

6. The extraction method according to claim 1, characterized in that: The Wash buffer solution consists of: 1M HEPES-KOH pH 7.5, 5M NaCl, 2M spermidine, and 1 tablet of Roche Complete Protease Inhibitor EDTA-Free.

7. The extraction method according to claim 1, characterized in that: In step (6), the centrifugation conditions are: 23°C, 150×g, 2 minutes, and the acceleration / deceleration rate is 2–3.

8. The extraction method according to claim 1, characterized in that: In step (6), the number of extracted cell nuclei is 2 × 10⁻⁶. 6 -3×10 6 One, suitable for subsequent CUT&Tag experiments.

9. A kit for extracting nuclear cells from Liriodendron tulipifera (Tulip Tree) cells, characterized in that: It includes the enzymatic hydrolysate, W5 solution, NE buffer, Wash buffer, and instructions for use as described in any one of claims 2-6.

10. The application of the *Liriodendron tulipifera* cell nuclei obtained by the method according to any one of claims 1-8 in CUT&Tag, characterized in that: Using the cell nucleus as the starting material, the protein-DNA interaction library was constructed by binding it with ConA magnetic beads and then incubating it with antibody-transposase.

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