A method for preparing Hi-C samples suitable for mollusc tissues

Abstract

This invention discloses a method for preparing Hi-C samples from mollusc tissues, belonging to the field of Hi-C technology. The method includes the following steps: cleaning and cutting the mollusc tissue to obtain tissue blocks; soaking the tissue blocks in a high-salt QN Buffer; placing the tissue blocks on a sterile filter membrane and cleaning them using the membrane's adsorption and friction; adding a CL Buffer containing formaldehyde, Triton X-100, and a protease inhibitor for cross-linking; terminating the cross-linking; homogenizing; and filtering to obtain the final sample. This invention effectively removes the mucus barrier through a unique pretreatment step and, combined with a dedicated cross-linking solution, achieves efficient and stable chromatin cross-linking, maximizing the preservation of nucleic acid integrity. This is of great significance for advancing three-dimensional genomics research on molluscs.

Description

Technical Field

[0001] This invention relates to the field of Hi-C technology, and more specifically to a method for preparing Hi-C samples suitable for mollusc tissues. Background Technology

[0002] Hi-C (High-throughput / resolution chromosome conformation capture) technology is a derivative of chromosome conformation capture (3C). It can capture high-resolution spatial interaction information between different gene loci across the entire genome, making it a key tool for studying three-dimensional genome structure. This technology uses formaldehyde fixation to cross-link spatially adjacent DNA fragments, followed by enzyme digestion, labeling, ligation, and high-throughput sequencing to elucidate chromatin interactions at the spatial level.

[0003] However, applying this technology to mollusc tissues (such as oysters, mussels, and scallops) faces significant challenges. First, these tissues secrete large amounts of polysaccharide-protein complex mucus, forming a physical barrier that severely hinders the penetration of formaldehyde molecules, resulting in low cross-linking efficiency. Second, and more critically, the activity of endogenous enzymes (such as nucleases) in mollusc tissues increases rapidly after in vitro examination, easily leading to DNA degradation and severely impacting the quality and reliability of subsequent Hi-C libraries. Traditional cross-linking methods and general pretreatment protocols are insufficient to effectively overcome the mucus barrier and rapidly inhibit endogenous enzyme activity, thus limiting the application of three-dimensional genomics in mollusc research.

[0004] Therefore, developing a pretreatment method that can efficiently remove mucus and rapidly achieve effective cross-linking to protect nucleic acid integrity is of vital importance for advancing three-dimensional genomics research in mollusks. Summary of the Invention

[0005] In view of the technical problems existing in the background art, the present invention provides a method for preparing Hi-C samples suitable for mollusc tissues. This method effectively removes the mucus barrier through a unique pretreatment step and, together with a special cross-linking solution, successfully achieves efficient and stable chromatin cross-linking of mollusc tissues, thus maximizing the preservation of nucleic acid integrity.

[0006] The present invention specifically adopts the following technical solution: A method for preparing Hi-C samples suitable for mollusc tissues includes the following steps: S1. Clean and cut the mollusc tissue to obtain tissue blocks; S2. After soaking the tissue block in QN Buffer, place the tissue block on a sterile filter membrane and clean the tissue block using the adsorption and friction of the filter membrane. The QN Buffer contains 2.5-10 M NaCl, 10-20 mM Tris-HCl, and 10-100 mM EDTA. S3. Add CL Buffer to the container holding the tissue block obtained in step S2 for cross-linking, wherein the CL Buffer contains 1-2.5% formaldehyde, 0.1-1.5% Triton X-100, 10-100mM EDTA, 1-10 mM protease inhibitor PMSF, 0.1-1% BSA and PBS at a final concentration. S4. After terminating the cross-linking, homogenize the mixture and filter it through a cell sieve to obtain the cell nuclear suspension for subsequent Hi-C library construction.

[0007] Preferably, the volume of the tissue block obtained in step S1 is 5-10 mm. 3 .

[0008] Preferably, the NaCl content in the QN Buffer is 5-7 M, with 5 M being optimal.

[0009] Preferably, the soaking conditions in step S2 with QN Buffer are 5-10 min at room temperature; the soaking time should not be too long, as excessive soaking time will cause the sample to dehydrate and the three-dimensional structure to change, thus affecting the experimental results.

[0010] Preferably, in step S2, the method of cleaning the tissue block by using the adsorption and friction of the filter membrane is as follows: wrap the tissue block with the filter membrane, gently scrape the filter membrane with the outside of the tweezers, and at the same time use absorbent paper to absorb the residual mucus on the back of the filter membrane where the tissue block is placed until there are no obvious water stains on the surface of the absorbent paper.

[0011] Preferably, the TritonX-100 content in the CL Buffer is 0.1-1.0%.

[0012] Preferably, in step S4, crosslinking is terminated using SC Buffer, which is a 0.1-0.5 mol glycine solution.

[0013] Preferably, in step S4, homogenization is performed using HB Buffer, which contains 10-20 mM Tris-HCl, 10-100 mM EDTA, 20-100 mM KCl, 0.1-0.5 M Surose, 0.1-1% Triton X-100, 20-100 mM Spermine, and 20-100 mM Spermindine.

[0014] Preferably, the crosslinking conditions in step S3 are: crosslinking in a vacuum environment for 15-45 min.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows: (1) Efficiently overcomes the mucus barrier: This invention uses a high-salt-concentration mucus-removing buffer QN Buffer for pretreatment, which can specifically and efficiently break down the mucus barrier unique to mollusc tissues, and significantly improve the penetration efficiency of subsequent cross-linking reagents.

[0016] (2) Effective protection of nucleic acid integrity: The method of the present invention is rapid and combined with cross-linking liquid CL Buffer containing enzyme inhibitors, which can quickly fix chromatin and inhibit active endogenous nucleases after in vitro, greatly reducing DNA degradation and ensuring the authenticity of Hi-C data.

[0017] (3) High crosslinking efficiency and quality: CL Buffer treatment in a vacuum environment ensures that the crosslinking reaction is more complete and uniform, thereby obtaining higher quality chromatin interaction data.

[0018] (4) Wide application and high reliability: The method of this invention is applicable to a variety of common mollusks (such as oysters, mussels, scallops, and sea snails), and has good universality and reproducibility, providing reliable technical support for three-dimensional genomics research of mollusks. Attached Figure Description

[0019] To more clearly illustrate the technical solution of the present invention, the accompanying drawings used in the present invention will be briefly described below. Obviously, the drawings described below are merely some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0020] Figure 1 The flowchart illustrates the method for preparing Hi-C samples suitable for mollusc tissues provided by this invention.

[0021] Figure 2The image shown is an agarose gel electrophoresis pattern from Example 3, where C1 is the group treated with 5M NaCl + 0.1% Triton X-100, C2 is the group treated with 5M NaCl + 0.5% Triton X-100, C3 is the group treated with 5M NaCl + 1.0% Triton X-100, and C4 is the group treated with 5M NaCl + 1.5% Triton X-100.

[0022] Figure 3 The image shows the agarose gel electrophoresis pattern from Example 4, where D1 is the group treated with 7M NaCl + 0.1% Triton X-100, D2 is the group treated with 7M NaCl + 0.5% Triton X-100, D3 is the group treated with 7M NaCl + 1.0% Triton X-100, and D4 is the group treated with 7M NaCl + 1.5% Triton X-100.

[0023] Figure 4 This is the agarose gel electrophoresis pattern from Example 5.

[0024] Figure 5 This is the agarose gel electrophoresis pattern from Example 6.

[0025] Figure 6 This is the agarose gel electrophoresis pattern from Comparative Example 1.

[0026] Figure 7 This is the agarose gel electrophoresis pattern of Comparative Example 2.

[0027] Figure 8 This is the agarose gel electrophoresis pattern of Comparative Example 3.

[0028] Figure 9 This is the agarose gel electrophoresis pattern of Comparative Example 4.

[0029] Figure 10 This is the agarose gel electrophoresis pattern of Comparative Example 5.

[0030] Figure 11 This is the agarose gel electrophoresis pattern of Comparative Example 6. Detailed Implementation

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains; the terminology used herein is for the purpose of describing particular implementations only and is not intended to limit the invention; the terms “comprising” and “having”, and any variations thereof, in the specification and claims of this invention are intended to cover non-exclusive inclusion.

[0032] To address the limitations of traditional cross-linking methods in Hi-C technology for mollusc tissues, this invention develops a Hi-C sample preparation method suitable for mollusc tissues. This method solves problems such as mucus physical barriers, low cross-linking efficiency, and easy DNA degradation, thereby promoting the application of Hi-C technology in the field of mollusc research.

[0033] like Figure 1 As shown, the method for preparing Hi-C samples suitable for mollusc tissues provided in this embodiment of the invention includes the following steps: (1) Clean and cut the mollusc tissue to obtain tissue blocks; (2) After soaking the tissue block in QN Buffer, place the tissue block on a sterile filter membrane and clean the tissue block by adsorption and friction of the filter membrane. The QN Buffer contains 2.5-10 M NaCl, 10-20 mM Tris-HCl and 10-100 mM EDTA. (3) Add CL Buffer to the container holding the tissue block for cross-linking, wherein the CL Buffer contains 1-2.5% formaldehyde (mass concentration), 0.1-1.5% Triton X-100 (mass concentration), 10-100mM EDTA, 1-10 mM protease inhibitor PMSF, 0.1-1% BSA (mass volume percentage) and PBS; (4) After terminating cross-linking, homogenize the mixture and filter it through a cell sieve to obtain the cell nucleus suspension for subsequent Hi-C library construction.

[0034] Further, in some embodiments, step (1) specifically involves: selecting fresh mollusc tissue and immediately placing it on ice to slow down enzyme activity; thoroughly rinsing the tissue surface with pre-cooled (4°C) PBS to remove mud, seaweed, and other attached substances; and then rapidly cutting the tissue into small pieces (approximately 5-10 mm³) on ice to increase the contact area of ​​subsequent reagents.

[0035] Furthermore, in some embodiments, the NaCl content in the QN Buffer is 5-7 M. When the cut tissue blocks are completely immersed in the QN Buffer, foam appears on the surface of the liquid during immersion, a characteristic of viscous liquids. The originally small pieces of tissue clump together. When the liquid around the tissue is stirred with a pipette tip, a gel-like, water-insoluble, transparent liquid is visible around the tissue. This invention denatures the glycoproteins in the mucus through salting out, effectively disrupting the polysaccharide-protein network structure of the mucus and significantly reducing its viscosity, thus clearing obstacles for the subsequent penetration of cross-linking reagents.

[0036] Furthermore, in some embodiments, the method of cleaning tissue blocks using the adsorption and friction of the filter membrane is as follows: fold the filter membrane to wrap the tissue, gently scrape the filter membrane with the outside of tweezers, and use the adsorption and friction of the filter membrane to remove the dissolved mucus. To remove the mucus as completely as possible, absorbent paper can be used to absorb any remaining mucus on the back of the filter membrane containing the sample until there are no obvious water stains on the surface of the absorbent paper. This invention, through soaking in QN Buffer combined with the adsorption and friction treatment of the filter membrane, can remove most of the mucus on the surface of the tissue block, allowing formaldehyde to fully penetrate into the sample tissue during cross-linking.

[0037] Furthermore, in some embodiments, the Triton X-100 content in the CL Buffer is 0.1-1.0%. The mucus secreted by mollusc tissues is rich in mucins, forming a dense network; Triton X-100 can partially disrupt these hydrophobic interactions and hydrogen bonds, reducing the viscosity of the mucus and allowing subsequent cross-linking solutions and reagents to effectively contact the tissue cells.

[0038] Furthermore, in some embodiments, the crosslinking is performed in a vacuum environment for 15-45 minutes. The vacuum environment helps the crosslinking agent to penetrate further into the tissue, improving crosslinking efficiency and uniformity.

[0039] Furthermore, in some embodiments, crosslinking is terminated using SC Buffer and homogenization is performed using HB Buffer, wherein SC Buffer is a 0.1-0.5 mol glycine solution and HB Buffer contains 10-20 mM Tris-HCl, 10-100 mM EDTA, 20-100 mM KCl, 0.1-0.5 M Surose, 0.1-1% Triton X-100, 20-100 mM Spermine, and 20-100 mM Spermindine.

[0040] The following are some specific embodiments. It should be noted that the embodiments described below are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. Where specific techniques or conditions are not specified in the embodiments, they shall be performed in accordance with the techniques or conditions described in the literature in this field or according to the product instructions. Reagents or instruments used, unless otherwise specified, are all conventional products that can be obtained commercially.

[0041] Example 1 Taking the clitellum garterii as an example, this example provides a method for preparing Hi-C samples suitable for mollusc tissues, including the following steps: (1) Tissue collection and preprocessing.

[0042] Select fresh adductor muscles from shellfish and immediately place them on ice for processing. Thoroughly rinse the surface of the adductor muscles with pre-cooled (4°C) PBS buffer to remove any adhering debris such as sand. Subsequently, rapidly cut the tissue into small pieces (approximately 5-10 mm) on ice. 3 ).

[0043] (2) Mucus removal treatment.

[0044] Prepare a high-salt-concentration mucus-removing buffer (QN Buffer) consisting of the following components: 5M NaCl, 10 mMtris-HCl, and 100 mM EDTA. Immerse the tissue blocks obtained in step (1) in the QN Buffer and soak at room temperature for 5 min.

[0045] After soaking, place the tissue block on a sterile filter membrane. Gently fold the membrane to enclose the tissue, and use forceps to gently scrape the membrane from the outside. Utilize the membrane's adsorption and friction to remove dissolved mucus. Simultaneously, use absorbent paper to blot away any remaining mucus from the back of the filter membrane containing the sample. Continue until the absorbent paper is free of visible water stains. Unfold the membrane and transfer the cleaned tissue block to a new centrifuge tube. Rinse the tissue block twice with pre-cooled 1×PBS. After rinsing, transfer the sample to a crosslinking tube and await crosslinking.

[0046] (3) Crosslinking treatment.

[0047] Prepare a cross-linking buffer (CL Buffer) consisting of the following components: 37% formaldehyde (final concentration 2%), Triton X-100 (0.1%), EDTA (10 mM), BSA (0.1%), protease inhibitors (PMSF: 0.1 mM; PI: 1X) and 1×PBS.

[0048] Add the above crosslinking solution to the crosslinking tube containing the tissue sample obtained in step (2), and place the crosslinking tube in a vacuum environment for 30 min for crosslinking.

[0049] (4) Termination of cross-linking and extraction of cell nuclei.

[0050] Prepare SC Buffer, which is a 0.3 mol glycine aqueous solution. Prepare homogenization buffer HB Buffer, which consists of the following components: Tris-HCl (10 mM), EDTA (10 mM), KCl (80 mM), Sucrose (0.5 M), Triton X-100 (0.5%), Spermine (5 mM), and Spermindine (5 mM).

[0051] After the cross-linking reaction in step (3) is completed, SC Buffer is added to terminate the cross-linking. Subsequently, HB Buffer is added and the tissue is gently homogenized using a handheld homogenizer to release the cell nuclei. After homogenization, the tissue is washed with HB Buffer and filtered through a cell sieve to obtain a high-quality cell nucleus suspension suitable for subsequent Hi-C library construction.

[0052] Example 2 Based on Example 1, this example verifies the effect of NaCl content on mucus removal by preparing QN Buffers with different salt concentrations. The specific experiments and results are as follows: First, tissue blocks were obtained according to step (1) of Example 1. Then, QN Buffers with NaCl contents of 1 M, 3 M, 5 M and 7 M were prepared respectively, and the other components and their contents were the same as in Example 1. Subsequently, the tissue blocks were immersed in different QN Buffers and soaked at room temperature for 5 min, with 3 replicates for each group.

[0053] After soaking, the tissue blocks were placed on a sterile filter membrane. The membrane was gently folded to wrap the tissue, and the outside of the membrane was gently scraped with forceps to remove the dissolved mucus through adsorption and friction. Simultaneously, absorbent paper was used to remove any remaining mucus from the back of the membrane containing the sample. Results showed that: in the 1 M NaCl treatment group, significant mucus adhered to the filter membrane, with noticeable stringy texture between tissues. Scraping the membrane revealed that the mucus was difficult to remove, leaving a considerable amount of residue. In the 3 M NaCl treatment group, the mucus was less than in the 1 M NaCl treatment group, but the stringy texture between tissues was still evident, and the mucus was difficult to remove when scraping the membrane. In the 5 M NaCl treatment group, the mucus contained a small amount of mucus, with less stringy texture than in the 3 M NaCl treatment group. The mucus was easier to remove when scraping the membrane, and the tissue was more dispersed after scraping. In the 7 M NaCl treatment group, the mucus contained a small amount of mucus, with some stringy texture between tissues, and almost no mucus remained after scraping. The above results show that the 1 M NaCl treatment group and the resulting samples still retained a large amount of mucus, which affected subsequent operations. In contrast, the samples obtained from the 5 M NaCl treatment group and the 7 M NaCl treatment group had less mucus residue. This demonstrates that the strategy of using high-salt-concentration QN Buffer combined with membrane adsorption and friction treatment can successfully remove the mucus barrier problem in mollusc tissues.

[0054] Example 3 Based on Example 1, this example investigated the effect of Triton X-100 content in the crosslinking solution CL Buffe on the crosslinking effect. The specific experiments and results are as follows: Steps (1) and (2) are completely consistent with step (1) of Example 1. Prepare CL Buffer with Triton X-100 content of 0.1%, 0.5%, 1.0% or 1.5%, and the other components and their contents are the same as in Example 1. Then, cross-linking treatment, termination of cross-linking and cell nucleus extraction are performed in sequence according to steps (3) and (4) of Example 1.

[0055] DNA extraction and detection were performed on the nuclear suspensions obtained from each treatment. The specific methods were as follows: the nuclear suspensions were centrifuged at 3500 g for 5 min at 4℃, the supernatant was discarded, and the precipitate was collected; the suspensions were resuspended in 1×PBS, and 100 μl of each suspension was transferred to 1.5 ml centrifuge tubes, labeled +K and -K respectively. A lysis system (mainly containing SDS and PK) was added to +K, while no lysis system was added to -K; the +K and -K samples were placed on a Thermo mixer and lysed overnight at 50℃ and 900 rpm; the remaining samples were transferred to new 1.5 ml tubes, centrifuged again at 3500 rpm and 4℃ for 5 min, the supernatant was discarded, the samples were flash-frozen in liquid nitrogen and then stored at -80℃; the overnight lysed samples were centrifuged at 12000 g for 2 min at room temperature, 100 μl of the supernatant was collected and purified with 1× magnetic beads; the purified DNA was detected by agarose gel electrophoresis.

[0056] Test results as follows Figure 2 As shown, the cross-linking status and genome integrity of the samples obtained under different Triton X-100 addition levels were relatively good. However, compared with C1-C3, the samples under C4 conditions showed a slight tailing, that is, the samples showed a slight degradation. This indicates that the cross-linking effect was better under medium and low concentrations of Triton X-100 than under high concentrations of Triton X-100.

[0057] Example 4 Compared to Example 3, the NaCl content in the QN Buffer used in step (2) is adjusted from 5 M to 7 M in this example, while other operations and detection methods are the same as in Example 3.

[0058] The test results in this example are as follows: Figure 3 As shown, slight trailing is visible under conditions D1 and D3, while D2 and D4 are relatively better, with D4 showing a slight trailing compared to D2.

[0059] contrast Figure 2 and Figure 3It can be seen that the gene integrity of the cross-linked sample obtained in Example 3 is better than that in this example. Higher concentrations of Triton X-100 and NaCl are not as effective as lower concentrations. The cross-linking effect is better when the NaCl concentration in QN Buffer is 5M and the Triton X-100 concentration in CL Buffer is 0.1%-1%.

[0060] Example 5 In this example, Hi-C samples were prepared using *Cynodon maxima*. The specific experiments and results are as follows: (1) Tissue collection and preprocessing.

[0061] Select fresh adductor muscles from shellfish and immediately place them on ice for processing. Rinse the surface of the adductor muscles thoroughly with pre-cooled (4°C) PBS buffer to remove any attached debris such as mud and sand. Then, quickly cut the tissue into small pieces (approximately 5-10 mm) on ice. 3 ).

[0062] (2) Mucus removal treatment.

[0063] Prepare a high-salt-concentration mucus-removing buffer, QN Buffer, with the same composition as in Example 1. Immerse the cut tissue block obtained in step (1) in QN Buffer and soak at room temperature for 5 min.

[0064] After soaking, place the tissue block on a sterile filter membrane. Gently fold the membrane to enclose the tissue, and use forceps to gently scrape the membrane from the outside. Utilize the membrane's adsorption and friction to remove dissolved mucus. Simultaneously, use absorbent paper to blot away any remaining mucus from the back of the filter membrane containing the sample. Continue until the absorbent paper is free of visible water stains. Unfold the membrane and transfer the cleaned tissue block to a new centrifuge tube. Rinse the tissue block twice with pre-cooled 1×PBS. After rinsing, transfer the sample to a crosslinking tube and await crosslinking.

[0065] (3) Crosslinking treatment.

[0066] Prepare a cross-linking buffer (CL Buffer) consisting of 37% formaldehyde (final concentration 2%), Triton X-100 (0.1%), EDTA (10 mM), BSA (0.1%), protease inhibitors (PMSF: 0.1 mM; PI: 1X) and 1×PBS.

[0067] Add the above crosslinking solution to the crosslinking tube containing the tissue sample obtained in step (2), and place the crosslinking tube in a vacuum environment for 30 min for crosslinking.

[0068] (4) Termination of cross-linking and extraction of cell nuclei.

[0069] Prepare SC Buffer, which is a 0.3 mol glycine aqueous solution. Prepare homogenization buffer HB Buffer, which consists of the following components: Tris-HCl (10 mM), EDTA (10 mM), KCl (80 mM), Sucrose (0.5 M), Triton X-100 (0.5%), Spermine (5 mM), and Spermindine (5 mM).

[0070] After the cross-linking reaction in step (3) is completed, SC Buffer is added to terminate the cross-linking. Subsequently, HB Buffer is added and the tissue is gently homogenized using a handheld homogenizer to release the cell nuclei. After homogenization, the tissue is washed with HB Buffer and filtered through a cell sieve to obtain a high-quality cell nucleus suspension suitable for subsequent Hi-C library construction.

[0071] (5) DNA extraction and detection.

[0072] Centrifuge the cell nucleus suspension at 3500 g for 5 min at 4 °C, discard the supernatant, and collect the precipitate. Resuspend the precipitate in 1×PBS, and transfer 100 μl of each to a 1.5 ml centrifuge tube, labeling them +K and -K respectively. Add lysis system (mainly containing SDS and PK) to the +K tube, but do not add lysis system to the -K tube. Place the +K and -K samples on a Thermo mixer and set the program to 50 °C and 900 rpm for overnight lysis. Transfer the remaining samples to a new 1.5 ml tube, set the centrifuge program to 3500 rpm and centrifuge again at 4 °C for 5 min. Discard the supernatant after centrifugation, flash freeze in liquid nitrogen, and then store in a -80 °C freezer. Centrifuge the overnight lysed samples at 12000 g for 2 min at room temperature, collect 100 μl of the supernatant, and add 1× magnetic beads for purification. Detect the purified DNA by agarose gel electrophoresis.

[0073] The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 4 As shown, the cross-linking state is good, the genome integrity is good, and there is basically no degradation.

[0074] Example 6 In this example, Hi-C samples were prepared using conch shells. The specific experiments and results are as follows: (1) Tissue collection and preprocessing.

[0075] Select the foot of a fresh conch and immediately place it on ice for processing; thoroughly rinse the surface of the foot with pre-cooled (4°C) PBS buffer to remove mud and other adhering substances, then quickly cut the tissue into small pieces (approximately 5-10 mm) on ice. 3 ).

[0076] (2) Mucus removal treatment.

[0077] Prepare a high-salt-concentration mucus-removing buffer, QN Buffer, with the same composition as in Example 1. Immerse the cut tissue block obtained in step (1) in QN Buffer and soak at room temperature for 5 min.

[0078] After soaking, place the tissue block on a sterile filter membrane. Gently fold the membrane to enclose the tissue, and use forceps to gently scrape the membrane from the outside. Utilize the membrane's adsorption and friction to remove dissolved mucus. Simultaneously, use absorbent paper to blot away any remaining mucus from the back of the filter membrane containing the sample. Continue until the absorbent paper is free of visible water stains. Unfold the membrane and transfer the cleaned tissue block to a new centrifuge tube. Rinse the tissue block twice with pre-cooled 1×PBS. After rinsing, transfer the sample to a crosslinking tube and await crosslinking.

[0079] (3) Crosslinking treatment.

[0080] Prepare a cross-linking buffer (CL Buffer) consisting of 37% formaldehyde (final concentration 2%), Triton X-100 (0.1%), EDTA (10 mM), BSA (0.1%), protease inhibitors (PMSF: 0.1 mM; PI: 1X) and 1×PBS.

[0081] Add the above crosslinking solution to the crosslinking tube containing the tissue sample obtained in step (2), and place the crosslinking tube in a vacuum environment for 30 min for crosslinking.

[0082] (4) Termination of cross-linking and extraction of cell nuclei.

[0083] Prepare SC Buffer, which consists of 0.3 mol glycine aqueous solution. Prepare homogenization buffer HB Buffer, which consists of Tris-HCl (10 mM), EDTA (10 mM), KCl (80 mM), Sucrose (0.5 M), Triton X-100 (0.5%), Spermine (5 mM), and Spermindine (5 mM).

[0084] After the cross-linking reaction in step (3) is completed, SC Buffer is added to terminate the cross-linking. Subsequently, HB Buffer is added and the tissue is gently homogenized using a handheld homogenizer to release the cell nuclei. After homogenization, the tissue is washed with HB Buffer and filtered through a cell sieve to obtain a high-quality cell nucleus suspension suitable for subsequent Hi-C library construction.

[0085] (5) DNA extraction and detection.

[0086] Centrifuge the cell nucleus suspension at 3500 g for 5 min at 4 °C, discard the supernatant, and collect the precipitate. Resuspend the precipitate in 1×PBS, and transfer 100 μl of each to a 1.5 ml centrifuge tube, labeling them +K and -K respectively. Add lysis system (mainly containing SDS and PK) to the +K tube, but do not add lysis system to the -K tube. Place the +K and -K samples on a Thermo mixer and set the program to 50 °C and 900 rpm for overnight lysis. Transfer the remaining samples to a new 1.5 ml tube, set the centrifuge program to 3500 rpm and centrifuge again at 4 °C for 5 min. Discard the supernatant after centrifugation, flash freeze in liquid nitrogen, and then store in a -80 °C freezer. Centrifuge the overnight lysed samples at 12000 g for 2 min at room temperature, collect 100 μl of the supernatant, and add 1× magnetic beads for purification. Detect the purified DNA by agarose gel electrophoresis.

[0087] The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 5 As shown, the cross-linking state is good, the genome integrity is good, and there is basically no degradation.

[0088] Comparative Example 1 Unlike Example 5, the preparation method of the Hi-C sample of *Cyprinus multinodosa* in this example includes the following steps: Step (1) is the same as step (1) in Example 5.

[0089] (2) Crosslinking treatment.

[0090] Prepare a crosslinking solution CL Buffer, with the same composition as in Example 5.

[0091] The tissue block obtained in step (1) is directly transferred to the crosslinking tube, the above crosslinking liquid is added to the crosslinking tube, and the crosslinking tube is placed in a vacuum environment for 30 min for crosslinking.

[0092] (3) Termination of cross-linking and extraction of cell nuclei.

[0093] This step is the same as step (4) in Example 5.

[0094] (4) DNA extraction and detection.

[0095] This step is the same as step (5) in Example 5.

[0096] The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 6 As shown, the shell adductor muscle of shellfish that has not undergone QN Buffer combined with membrane adsorption and friction treatment showed severe degradation and poor genome integrity after cross-linking.

[0097] Comparative Example 2 Unlike Example 5, the preparation method of the Hi-C sample of *Cyprinus multinodosa* in this example includes the following steps: Step (1) is the same as step (1) in Example 5.

[0098] (2) Mucus removal treatment.

[0099] Prepare a high-salt-concentration desmear buffer QN Buffer with the same composition as in Example 1.

[0100] The tissue block obtained in step (1) was immersed in QN Buffer and soaked at room temperature for 5 min. The soaked tissue block was directly transferred to the crosslinking tube using a pipette tip. In this example, no filter membrane wrapping, scraping, or back-side suction was performed on the tissue block.

[0101] Steps (3), (4), and (5) are the same as steps (3), (4), and (5) in Example 5, respectively. The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 7 As shown, the shell adductor muscle of shellfish that has been soaked in QN Buffer but not subjected to filter membrane adsorption and friction treatment exhibits moderate degradation and poor genome integrity in the samples obtained after cross-linking.

[0102] Comparative Example 3 Unlike Example 5, the preparation method of the Hi-C sample of *Cyprinus multinodosa* in this example includes the following steps: Steps (1) and (2) are the same as steps (1) and (2) in Example 5.

[0103] (3) Crosslinking treatment.

[0104] The cross-linking solution was prepared with the following components: 37% formaldehyde (final concentration 2%), Triton X-100 (0.1%), EDTA (10 mM), BSA (0.1%), protease inhibitors (PMSF: 0.1 mM; PI: 1X) and 1×PBS.

[0105] Add the above crosslinking solution to the crosslinking tube containing the tissue sample obtained in step (2), and place the crosslinking tube in a vacuum environment for 30 min for crosslinking.

[0106] Steps (4) and (5) are the same as steps (4) and (5) in Example 5.

[0107] The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 8 As shown, removing Triton X-100 from the crosslinking solution leads to slight degradation of the crosslinked sample, with slight tailing of the crosslinked strips and poor genome integrity. However, the sample still shows a clear main band, which also demonstrates the positive effect of QN Buffer combined with membrane adsorption and friction treatment on maintaining genome integrity after crosslinking.

[0108] Comparative Example 4 Unlike Example 6, the preparation method of the Hi-C sample of conch in this example includes the following steps: Step (1) is the same as step (1) in Example 6.

[0109] (2) Crosslinking treatment.

[0110] Prepare a crosslinking solution CL Buffer, with the same composition as in Example 6.

[0111] The tissue block obtained in step (1) is directly transferred to the crosslinking tube, the above crosslinking liquid is added to the crosslinking tube, and the crosslinking tube is placed in a vacuum environment for 30 min for crosslinking.

[0112] (3) Termination of cross-linking and extraction of cell nuclei.

[0113] This step is the same as step (4) in Example 6.

[0114] (4) DNA extraction and detection.

[0115] This step is the same as step (5) in Example 6.

[0116] The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 9 As shown, the samples of snail feet that have not undergone QN Buffer combined with membrane adsorption and friction treatment showed degradation and poor genome integrity after cross-linking.

[0117] Comparative Example 5 Unlike Example 6, the preparation method of the Hi-C sample of conch in this example includes the following steps: Step (1) is the same as step (1) in Example 6.

[0118] (2) Mucus removal treatment.

[0119] Prepare a high-salt-concentration desmear buffer QN Buffer with the same composition as in Example 1.

[0120] The tissue block obtained in step (1) was immersed in QN Buffer and soaked at room temperature for 5 min. The soaked tissue block was directly transferred to the crosslinking tube using a pipette tip. In this example, no filter membrane wrapping, scraping, or back-side suction was performed on the tissue block.

[0121] Steps (3), (4), and (5) are the same as steps (3), (4), and (5) in Example 6. The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 10As shown, the abdominal foot that was not subjected to filter adsorption and friction treatment was moderately degraded after cross-linking, and the genome integrity was poor; however, compared with Comparative Example 4, the sample was found to have a slight main band, indicating that the genome integrity was significantly improved compared with Comparative Example 4.

[0122] Comparative Example 6 Unlike Example 6, the preparation method of the Hi-C sample of conch in this example includes the following steps: Steps (1) and (2) are the same as steps (1) and (2) in Example 6.

[0123] (3) Crosslinking treatment.

[0124] The cross-linking solution was prepared with the following components: 37% formaldehyde (final concentration 2%), Triton X-100 (0.1%), EDTA (10 mM), BSA (0.1%), protease inhibitors (PMSF: 0.1 mM; PI: 1X) and 1×PBS.

[0125] Add the above crosslinking solution to the crosslinking tube containing the tissue sample obtained in step (2), and place the crosslinking tube in a vacuum environment for 30 min for crosslinking.

[0126] Steps (4) and (5) are the same as steps (4) and (5) in Example 6.

[0127] The results of the purified DNA agarose gel electrophoresis quality control are as follows: Figure 11 As shown, removing Triton X-100 from the crosslinking solution results in a slight tailing of the crosslinked strips and poor genome integrity, but the sample still shows a clear main band.

[0128] In summary, this invention effectively removes the mucus barrier through a unique pretreatment step, and, in conjunction with a dedicated cross-linking solution, successfully achieves efficient and stable chromatin cross-linking, thus maximizing the preservation of nucleic acid integrity in mollusc tissues.

[0129] It should be noted that the present invention is not limited to the above-described embodiments. The above embodiments are merely examples, and any embodiments that have the same structure and perform the same effects as the technical concept within the scope of the present invention are included within the scope of the present invention. Furthermore, various modifications that can be conceived by those skilled in the art to the embodiments, and other ways of constructing by combining some of the constituent elements of the embodiments, without departing from the spirit of the present invention, are also included within the scope of the present invention.

Claims

1. A method for preparing Hi-C samples suitable for mollusc tissues, characterized in that, Includes the following steps: S1. Clean and cut the mollusc tissue to obtain tissue blocks; S2. After soaking the tissue block in QN Buffer, place the tissue block on a sterile filter membrane and clean the tissue block using the adsorption and friction of the filter membrane. The QN Buffer contains 2.5-10 M NaCl, 10-20 mM Tris-HCl, and 10-100 mM EDTA. S3. Add CL Buffer to the container holding the tissue block obtained in step S2 for cross-linking. The CL Buffer contains 1-2.5% formaldehyde, 0.1-1.5% Triton X-100, 10-100 mM EDTA, 1-10 mM protease inhibitor PMSF, 0.1-1% BSA and PBS at a final concentration. S4. After terminating the cross-linking, homogenize the mixture and filter it through a cell sieve to obtain the cell nuclear suspension for subsequent Hi-C library construction.

2. The method for preparing Hi-C samples according to claim 1, characterized in that, The volume of the tissue block in step S1 is 5-10 mm. 3 .

3. The method for preparing Hi-C samples according to claim 1, characterized in that, The QN Buffer contains 5-7 M NaCl.

4. The method for preparing Hi-C samples according to claim 3, characterized in that, The QN Buffer contains 5 M NaCl.

5. The method for preparing Hi-C samples according to claim 1, characterized in that, In step S2, the soaking time is 5-10 minutes.

6. The method for preparing Hi-C samples according to claim 1, characterized in that, In step S2, the method of cleaning the tissue block by using the adsorption and friction of the filter membrane is as follows: wrap the tissue block with the filter membrane, gently scrape the filter membrane with the outside of the tweezers, and at the same time use absorbent paper to absorb the residual mucus on the back of the filter membrane where the tissue block is placed until there are no obvious water stains on the surface of the absorbent paper.

7. The method for preparing Hi-C samples according to claim 1, characterized in that, The TritonX-100 content in the CL Buffer is 0.1-1.0%.

8. The method for preparing Hi-C samples according to claim 1, characterized in that, In step S4, crosslinking is terminated using SCBuffer, which is a 0.1-0.5 mol glycine solution.

9. The method for preparing Hi-C samples according to claim 1, characterized in that, In step S4, homogenization is performed using HBBuffer, which contains 10-20 mM Tris-HCl, 10-100 mM EDTA, 20-100 mM KCl, 0.1-0.5 M Surose, 0.1-1% Triton X-100, 20-100 mM Spermine, and 20-100 mM M Permindine.

10. The method for preparing Hi-C samples according to claim 1, characterized in that, In step S3, crosslinking is performed in a vacuum environment for 15-45 minutes.

Images