CUTTag library building method for frozen tissues

Abstract

The embodiment of the invention discloses a CUAmp of a frozen tissue. The invention discloses a Tag database building method. The method comprises the steps of core extraction, formaldehyde crosslinking and crosslinking termination, carrying out incubation on the cell nucleus and ConABeads, carrying out primary antibody incubation, and carrying out secondary antibody incubation; incubating the Tn5 enzyme, and breaking the Tn5 enzyme; carrying out decrosslinking; dNA extraction, PCR amplification and purification; and determining the library concentration. According to the invention, formaldehyde decrosslinking conditions are researched, and the frozen tissue CUTamp is provided; the Tag database building method has the advantages of high sequencing depth and coverage, high quality, high number of peaks, low background noise, high repeatability and high specificity, so that the reliability and scientific value of data are improved.

Description

Technical Field

[0001] Embodiments of the present invention relate to the field of biotechnology, and specifically to a method for constructing a CUT&Tag library for frozen tissues. Background Art

[0002] CUT&Tag is a newer generation of technology for detecting the interaction between histones (or other proteins such as transcription factors) and DNA following the ChIP technology. It mainly uses antibodies to target the target protein, and then a fusion protein (such as pA-Tn5) is used to cut and tag the DNA region bound by the target protein. Subsequently, high-throughput sequencing is used to identify the DNA sites bound by the protein, which has advantages such as high resolution, low background, and high sensitivity.

[0003] Mammalian living cells have a complete cell structure and physiological state, which can ensure the normal interaction between the target protein and DNA. In living cells, the localization, conformation of proteins, and their binding patterns with DNA are all in a natural state, and the CUT&Tag technology can well capture these real interaction situations. In addition, the CUT&Tag technology relies on the activity of enzymes (such as Tn5 transposase) to perform DNA cutting and tagging operations. Mammalian living cells provide a suitable physiological environment, including an appropriate temperature (around 37°C), pH value (about 7.4), and ion concentration, etc., which can ensure that the enzyme exhibits its best activity. Under such conditions, Tn5 transposase can work efficiently and accurately operate in the target protein-DNA binding region.

[0004] The materials in the laboratory are generally frozen biological samples. The freezing process will cause the formation of ice crystals inside the cells, and the growth of ice crystals will lead to the destruction of the cell structure. The cell membrane ruptures and the cell organelles are damaged, which will cause changes in the localization and distribution of proteins. Proteins that were originally tightly bound to DNA may shift or denature due to the destruction of the cell structure, and cannot truly reflect their binding state in living cells. In addition, the temperature of frozen tissues is much lower than the temperature at which the enzyme exhibits its best activity. Low temperature will inhibit the activity of the enzyme. During the thawing process, the melting of ice crystals in the tissue will cause a drastic change in the local environment, which will also have an adverse effect on the activity of the enzyme, resulting in the inability of the CUT&Tag technology to operate normally or produce inaccurate results. Summary of the Invention

[0005] For this reason, embodiments of the present invention provide a method for constructing a CUT&Tag library for frozen tissues.

[0006] In order to achieve the above object, embodiments of the present invention provide the following technical solutions:

[0007] A method for constructing a CUT&Tag library for frozen tissues, the method comprising:

[0008] (1) Nuclear extraction, formaldehyde cross-linking, termination of cross-linking;

[0009] (2) Incubation of cell nuclei with ConABeads, incubation with primary antibody, incubation with secondary antibody;

[0010] (3) Incubation with Tn5 enzyme, fragmentation by Tn5 enzyme;

[0011] (4) Decross-linking;

[0012] (5) DNA extraction, PCR amplification and purification;

[0013] (6) Determination of library concentration.

[0014] Further, the lysis solution used in the nuclear extraction step comprises a lysis buffer and a lysis dilution buffer with a volume ratio of 1:1. Among them,

[0015] The composition of the lysis buffer is: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.10% Tween-20, 0.10% Nonidet P40 Substitute, 0.01% Digitonin, 0.10% BSA, 1 mM DTT;

[0016] The composition of the lysis dilution buffer is: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.10% BSA, 1 mM DTT.

[0017] Further, the conditions for formaldehyde cross-linking are: 0.1% formaldehyde, room temperature, 2 min;

[0018] The conditions for terminating cross-linking are: 0.2% glycine, room temperature, 5 min.

[0019] Further, the conditions for incubating cell nuclei with ConABeads are: room temperature, 10 min;

[0020] The conditions for incubating with the primary antibody are: standing overnight at 4 °C, with mixing 3 - 4 times during this period;

[0021] The conditions for incubating with the secondary antibody are: rotary incubation, with mixing once during this period.

[0022] Further, the conditions for incubating with Tn5 enzyme are: rotary incubation, with mixing once during this period;

[0023] The conditions for fragmentation by Tn5 enzyme are: 37 °C, 1 h, with mixing once during this period.

[0024] Further, the conditions for de-crosslinking are: adding Proteinase K and incubating at 60 °C for 60 min.

[0025] Further, the frozen tissue is a tissue or organ of a mammal, and the mammals include humans, macaques, mice, and canines, and the tissue or organ includes the brain, heart, liver, spleen, lung, kidney, muscle, and intestine.

[0026] The embodiments of the present invention have the following advantages:

[0027] The present invention studies the conditions for formaldehyde de-crosslinking. The provided method for constructing a library of frozen tissue CUT&Tag has high sequencing depth and coverage, high-quality and high-number peaks, low background noise, high repeatability and specificity, thereby improving the reliability and scientific value of the data. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the drawings in the following description are only exemplary, and for those of ordinary skill in the art, other implementation drawings can be obtained according to the provided drawings without creative efforts.

[0029] Figure 1 It is a comparison result of three different library construction methods provided by the present invention under the H3K4me3 label;

[0030] Figure 2 It is a comparison result of three different library construction methods provided by the present invention under the H3K27ac label;

[0031] Figure 3 It is a comparison result of the present invention with public data;

[0032] Figure 4 It is a comparison result of the present invention with public data;

[0033] Figure 5 It is a comparison result of the present invention with the theoretical distribution. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0034] The following specific embodiments illustrate the implementation manners of the present invention. Those familiar with this technology can easily understand other advantages and effects of the present invention from the content disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0035] Prepare reagents:

[0036] (1) Nuclear extraction reagent

[0037] Composition of washing buffer: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.10% BSA, 0.10% Tween-20, 1 mM DTT, with solvent being nuclease-free water;

[0038] Composition of lysis buffer: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.10% Tween-20, 0.10% Nonidet P40 Substitute, 0.01% Digitonin, 0.10% BSA, 1 mM DTT, with solvent being nuclease-free water;

[0039] Composition of lysis dilution buffer: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl 2 , 0.10% BSA, 1 mM DTT, with solvent being nuclease-free water.

[0040] (2) Formaldehyde cross-linking reagent

[0041] 1x TTBL: Take 2 mL of 5×TTBL and add 8 mL of water;

[0042] 2.5 M glycine solution: 0.9375 g of glycine powder, make up the volume to 5 mL with water;

[0043] Aliquot 5 mL of PBS solution and 5 mL of water respectively, and pre-cool on ice.

[0044] (3) CUT&Tag reagent (60 libraries)

[0045] 1×Binding Buffer: Take 2500 μL of 10×Binding Buffer and add 22500 μL of ddH 2 O;

[0046] AntibodyBuffer: Take 4000 μL of AntibodyBuffer(-) and add 40 μL of 5% Digitonin;

[0047] Wash Buffer: Take 1000 μL of 10×Wash Buffer, add 100 μL of 100×PIC, and then add 8900 μL of ddH 2 O.

[0048] Example 1

[0049] A CUT&Tag Library Construction Method for Frozen Tissues

[0050] (1) Nucleus Extraction Steps

[0051] 1.1 Tissue Equilibration: Put the tissue into the cryostat for equilibration more than 2 hours in advance. If the experiment starts early, it can be put in for equilibration overnight; Mortars, spatulas, etc. needed for liquid nitrogen grinding need to be sterilized one day in advance and placed in the oven overnight for drying;

[0052] 1.2 Prepare the Tissue: Fix the equilibrated tissue on the sample holder with OCT, check the cryostat, sample box, mortar and reagents, etc., and prepare for sectioning;

[0053] 1.3 Sectioning: After waiting for the OCT to completely solidify (about 5 minutes), fix the sample holder and start sectioning. The section thickness is 50 - 70 μm. The cut tissue is collected into the corresponding sample box and can be temporarily stored in the cryostat;

[0054] 1.4 Liquid Nitrogen Grinding: First pre-cool the mortar, grinding rod and spatula with liquid nitrogen, then pour the tissue into the liquid nitrogen and quickly grind with the mortar rod. Pay attention to replenishing the liquid nitrogen in time when it is exhausted (to avoid the tissue being exposed to air for a long time) until the tissue is ground into a fine powder without particles;

[0055] 1.5 Lysis: Transfer the above tissue powder to a pre-cooled glass mortar, add 2 mL of freshly prepared lysis buffer with a volume ratio of 1:1 of lysis buffer and lysis dilution buffer. First grind with rod A on ice for 20 times, then grind with rod B for 20 times, and lyse on ice for 5 minutes;

[0056] 1.6 Filtration: Place a 40 μm sieve on a pre-cooled 50 mL centrifuge tube, and transfer the lysed tissue fluid to the sieve for filtration. If the tissue fluid is too viscous, 2 - 4 mL of lysis buffer can be added for dilution;

[0057] 1.7 Collect the Nuclei: Aliquot the above tissue fluid into 1.5 mL centrifuge tubes, centrifuge at 500 xg at 4°C for 5 minutes, and discard the supernatant completely;

[0058] 1.8 Wash with Lysis Buffer Once: Resuspend the nuclei in the precipitate with freshly prepared lysis buffer, centrifuge at 500 xg at 4°C for 5 minutes, and discard the supernatant completely.

[0059] (2) Formaldehyde Crosslinking and Crosslinking Termination Steps

[0060] 2.1 Wash Twice with Wash Buffer: Resuspend the nuclei in the precipitate with freshly prepared Wash Buffer, centrifuge at 500 xg at 4°C for 5 minutes, and discard the supernatant completely; Repeat once.

[0061] 2.2 Formaldehyde micro-crosslinking: Resuspend the cells with 1 mL of pre-cooled PBS, let it stand at room temperature for 10 min (wait for the nuclear suspension to return to room temperature), then add 2.7 μL of 37% paraformaldehyde to each mL of the nuclear suspension, shake for 30 s, and place at room temperature for 2 min (shake once in the middle);

[0062] 2.3 Terminate crosslinking: Add 30 μL of freshly prepared 2.5 M glycine to each mL of the crosslinked nuclear suspension, place at room temperature for 5 min (shake 2 - 3 times during this period);

[0063] 2.4 Collect nuclei: Put the above nuclear suspension into a centrifuge, centrifuge at 4 °C, 1300 xg for 4 min, and discard all the supernatant;

[0064] 2.5 Wash once with Wash Buffer: Resuspend the nuclei in the precipitate with freshly prepared Wash Buffer, centrifuge at 4 °C, 500 xg for 5 min, and discard all the supernatant;

[0065] 2.6 Resuspend nuclei: Resuspend the nuclei from multiple tubes of the same tissue in a 1.5 mL centrifuge tube with freshly prepared 1x TTBL, and pipette up and down thoroughly to mix;

[0066] 2.7 Nucleus counting: Pipette 18 μL from the above nuclear suspension into a PCR tube, add 2 μL of AO / PI fluorescent dye, pipette up and down thoroughly to mix, then take 10 μL and add it to a disposable cell counting chamber for counting on the machine. Count 3 - 4 fields of view for each tissue, and a cell density of 2500 - 6500 cells / μL is more appropriate.

[0067] Note: 1. During cell counting, the resuspended cell suspension should be placed on ice;

[0068] 2. When it is found that the nucleus concentration is too high during counting, it can be appropriately diluted with freshly prepared 1x TTBL;

[0069] 3. When it is found that the nuclei are not clean enough, they can be washed several more times with freshly prepared Wash Buffer;

[0070] 4. During the CUT&Tag experiment, ConABeads will specifically adsorb nuclei, and almost all impurities in the nuclear suspension can be filtered out during this process. Therefore, the requirement for the cleanliness of nuclei in the CUT&Tag experiment is much lower than that in the ATAC experiment.

[0071] Steps (3) - (6) are carried out according to the content in the instruction manual of the TD903 kit produced by Nanjing Novoprotein Biological Co., Ltd.:

[0072] (3) Incubate cells (nuclei) with ConABeads.

[0073] (4) Primary antibody incubation.

[0074] (5) Secondary antibody incubation.

[0075] (6) pA / G-Tnp incubation.

[0076] (7) Fragmentation

[0077] 7.1 Take 40 μL of Dig-300 Buffer, add 10 μL of 5×TTBL, and mix well.

[0078] 7.2 Take the eight-strip tube from the previous step, centrifuge briefly to collect the reaction solution, place the eight-strip tube on the magnetic rack, and discard the supernatant completely after the liquid becomes clear (30 sec - 2 min).

[0079] 7.3 Add 50 μL of diluted TTBL to each sample and mix well.

[0080] 7.4 Place the eight-strip tube in a PCR instrument and incubate at 60 °C for 10 min, 60 min, and 12 h respectively. Invert and mix once after 30 min.

[0081] Note: The thermal lid of the PCR instrument is at 50 °C. The PCR instrument needs to be turned on in advance and the program needs to be run.

[0082] Steps (8) - (10) are carried out according to the content in the instruction manual of the TD903 kit produced by Nanjing Novoprotein Biological Co., Ltd.:

[0083] (8) DNA extraction.

[0084] (9) Library amplification.

[0085] (10) Purification of PCR products.

[0086] When the inventor was constructing a library from frozen tissues, after nuclear extraction, the formaldehyde cross-linking method recommended by the Steven HenikoffLab was used, that is, the de-cross-linking step was not set. As a result, it was found that although the library was significantly enriched at the TSS site, the enrichment degree of the library was not high, and various indicators such as the number of peaks and FRiP did not reach the passing level. Therefore, the present invention adds a de-cross-linking step. Specifically, three de-cross-linking gradients are set, namely 10 min, 1 h, and 12 h (overnight). The index statistics are shown in Table 1 below, and the accompanying drawings more intuitively show the differences among these gradients: overnight de-cross-linking is obviously over-de-cross-linked and the library construction fails. The de-cross-linking time of 1 h is significantly better than that of 10 min. Therefore, it is determined that the de-cross-linking condition of 60 °C for 1 h is the optimal solution. On this basis, the present invention compares the data generated by using this library construction method with the public data, and it can be clearly seen that the enrichment degree of the data obtained according to the method of the present invention is much higher than the published public data (Figure 3 ), while taking into account the advantage of low background noise Figure 4 ), especially in the Fingerprint metric, the data has basically reached the most ideal distribution Figure 5 ).

[0087] Table 1

[0088]

[0089] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it on the basis of the present invention, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention fall within the scope of the present invention claimed.

Claims

1. A CUT&Tag library construction method for frozen tissue, characterized in that: The method comprises: (1) core extraction, formaldehyde cross-linking, and cross-linking termination; (2) incubation of cell nuclei with ConABeads, primary antibody, and secondary antibody; (3) Tn5 enzyme incubation, Tn5 enzyme interruption; (4) Decrosslinking; (5) DNA extraction, PCR amplification and purification; (6) Determination of library concentration.

2. The CUT&Tag library construction method for frozen tissue according to claim 1, characterized in that: The lysis solution used in the nuclear extraction step includes a lysis buffer and a lysis dilution buffer in a volume ratio of 1:1, wherein: The composition of the lysis buffer was: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.10% Tween-20, 0.10% Nonidet P40 Substitute, 0.01% Digitonin, 0.10% BSA, 1 mM DTT; The composition of the lysis dilution buffer is: 10 mM, pH 7.4 Tris-HCl, 10 mM NaCl, 3 mM MgCl2, 0.10% BSA, 1 mM DTT.

3. The CUT&Tag library construction method for frozen tissue according to claim 1, characterized in that: The formaldehyde cross-linking conditions are: 0.1% formaldehyde, room temperature, 2 min; The conditions for terminating cross-linking are: 0.2% glycine, room temperature, 5 min.

4. The CUT&Tag library construction method for frozen tissue according to claim 1, characterized in that: The conditions for incubating the cell nuclei with ConABeads were: room temperature, 10 min; The conditions for the primary antibody incubation are: standing at 4°C overnight, mixing 3-4 times during the period; The conditions for the secondary antibody incubation are: rotating incubation, mixing once during the incubation.

5. The CUT&Tag library construction method for frozen tissue according to claim 1, characterized in that: The conditions for the Tn5 enzyme incubation are: rotating incubation, mixing once during the process; The conditions for Tn5 enzyme disruption are: 37° C., 1 h, with mixing once during the period.

6. The CUT&Tag library construction method for frozen tissue according to claim 1, characterized in that: The conditions for de-crosslinking are: adding proteinase K and incubating at 60° C. for 60 min.

7. The CUT&Tag library construction method for frozen tissue according to claim 1, characterized in that: The frozen tissue is a tissue organ of a mammal, including humans, macaques, mice, and dogs, and the tissue organ includes the brain, heart, liver, spleen, lung, kidney, muscle, and intestine.

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