Preparation method and application of animal cell nucleus suspension

文档序号：712516 发布日期：2021-04-16 浏览：6次中文

阅读说明：本技术 一种动物细胞核悬液的制备方法及其应用 (Preparation method and application of animal cell nucleus suspension ) 是由周煌凯高川陈飞钦夏昊强徐毓璇梁国霞于 2020-12-31 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种动物细胞核悬液的制备方法及其应用。本发明提供的制备方法包括组织处理破碎、重悬、离心、沉淀、重悬、细胞筛过滤、重悬离心,最后检测核浓度。本发明提供的动物细胞核悬液满足各类高通量单细胞测序平台的细胞悬液质量要求,有利于其进一步的推广应用。(The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of an animal cell nucleus suspension. The preparation method provided by the invention comprises the steps of tissue treatment and crushing, heavy suspension, centrifugation, precipitation, heavy suspension, cell sieve filtration, heavy suspension and centrifugation, and finally nuclear concentration detection. The animal cell nucleus suspension provided by the invention meets the cell suspension quality requirements of various high-throughput single cell sequencing platforms, and is favorable for further popularization and application.)

1. A method for preparing a cell nucleus suspension, which is characterized by comprising the following steps:

s1, taking 45-55 mg of target tissue, adding 450-550 mu L of lysis solution into the target tissue, standing the tissue on ice for 4-6 min, and shearing the tissue into 1-3 mm³Size fraction to obtain tissue fraction containing lysate;

s2, transferring the tissue fragments containing the lysate prepared in the step S1 to a grinding tube, quickly pressing for 1-2 times, and standing on ice for 3-5 min to prepare a tissue fluid;

s3, pressing the tissue fluid prepared in the step S2 for 8-12 times, standing on ice for 2-3 min, pressing again for 14-16 times, and quickly transferring to a new centrifugal tube to prepare a homogenate mixed solution;

s4, adding 450-550 mu L of heavy suspension Buffer into the homogenate mixed liquor prepared in the step S3, reversing the mixture back and forth for 5-8 times, mixing the mixture evenly, then centrifuging the mixture, and transferring the supernatant into a 15mL centrifuge tube to prepare new homogenate;

s5, centrifuging the new homogenate prepared in the step S4, removing supernatant, adding 1-2 mL of resuspension buffer to fully resuspend and precipitate, continuously repeating for 2 times, then adding iodixanol, blowing, uniformly mixing, adding 700 mu L of resuspension buffer, centrifuging again, removing impurities and supernatant by using a pipette, and keeping lower-layer liquid;

s6, adding 1-2 mL of resuspension buffer into the lower layer liquid in the step S5, blowing and sucking gently to mix uniformly, then centrifuging to remove supernatant, finally leaving 180-220 mu L of resuspension buffer in a centrifugal tube, adding 450-550 mu L of resuspension buffer into the supernatant, and blowing and sucking gently to resuspend to prepare cell suspension;

s7, filtering the cell suspension prepared in the step S6 by using a cell sieve, adding 450-550 mu L of heavy suspension buffer into the cell suspension, centrifuging, collecting precipitates, then cleaning the precipitates for 2 times by using a sterilizing solution, centrifuging, removing supernate, adding precooled 1 XPBS into the precipitates, and detecting the concentration of the precooled 1 XPBS to obtain the cell suspension.

2. The method of claim 1, wherein the lysis solution of steps S1 and S2 comprises sucrose with a molar mass of 250mM, Tris-HCl with a molar mass of 50mM, CaCl with a molar mass of 50mM₂MgCl with a molar mass of 50mM₂DTT with the molar mass of 1mM, CA-630 with the mass percent of 0.1%, RNase Inhibitor with the unit of enzyme activity of 0.4U/. mu.L, BSA with the mass concentration of 0.04% and 1 × protease Inhibitor.

3. The method of claim 1, wherein the resuspension buffer in steps S4, S5, S6 and S7 is 1 XPBS containing 0.04% BSA by mass, 0.35M mannitol by final concentration, and 0.2U/. mu.L RNase Inhibitor activity.

4. The method according to claim 1, wherein the centrifugation step in step S4 is performed at 1600-1700 rpm for 8-12S.

5. The method according to claim 1, wherein the centrifugation step of step S5 is performed at 2600 to 2700rpm for 4 to 6 min; the mass percent of the iodixanol is 55-65%, and the adding amount is 0.6-0.8 time of the total volume of the liquid; and the rotating speed during re-centrifugation is 10000-12000 rpm, and the centrifugation time is 15-20 min.

6. The method according to claim 1, wherein the centrifugation step in step S6 is performed at 2600-2700 rpm for 5-8 min.

7. The method according to claim 1, wherein the centrifugation step of step S7 is performed at 2600 to 2700rpm for 4 to 7 min; the sterilizing liquid comprises the following components in parts by weight: 5-10 parts of honeysuckle powder, 3-5 parts of allicin, 2-3 parts of fructus forsythiae extract and 50-60 parts of deionized water.

8. Use of the preparation method of claim 1 in the construction of a single-cell transcriptome sequencing library.

9. The use of claim 8, wherein the nuclear suspension prepared by the method is placed on ice and the pool is built up for 30 min.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a preparation method and application of an animal cell nucleus suspension.

Background

The nucleus is the largest and most important cell structure in eukaryotic cells, is the control center of cytogenetics and metabolism, and is one of the most significant marks for distinguishing eukaryotic cells from prokaryotic cells. The nucleus plays an important role in the metabolism, growth and differentiation of cells, and is the main site where genetic material exists. Although the shape of the nucleus varies, its basic structure is approximately the same, and is composed mainly of nuclear envelope, chromatin, nuclear skeleton, nucleoli and nucleome.

In the prior art, no special method for preparing the suspension of the animal cell nucleus is found, and the suspension of the animal cell nucleus is generally prepared directly, because the animal cell nucleus contains various structures such as chromatin, nucleoli, nucleosomes and the like and also contains genetic material DNA, in the actual preparation process of the suspension of the cell nucleus, how to ensure the integrity of the materials is not found in the prior art. However, in the next generation of high throughput single cell sequencing platform, there are cases where sequencing can be performed only by preparing tissues into cell nucleus suspensions, such as tissues with excessive cell volume, tissues with high energy consumption and multiple mitochondria, and the like.

The known cell nucleus suspensions in the prior art are all prepared by adopting plant cell nuclei, for example, Chinese patent CN103776678B discloses a preparation method of strawberry cell nucleus suspension, Chinese patent CN103940646B discloses a preparation method of anthurium andraeanum cell nucleus suspension, and Chinese patent application CN103940656B discloses a preparation method of usnea pineapple cell nucleus suspension.

In view of the above, there is a need in the market for a technology that can efficiently prepare suspensions of animal cell nuclei.

Disclosure of Invention

Aiming at the defects generally existing in the prior art, the invention creatively provides a preparation method of an animal cell nucleus suspension and application thereof. The animal cell nucleus suspension prepared by the invention can be used for constructing a single-cell transcriptome sequencing library, and is beneficial to further popularization and application.

In order to achieve the purpose, the invention adopts the technical scheme that:

a method for preparing a cell nucleus suspension comprises the following steps:

s2, transferring the tissue fragments containing the lysate prepared in the step S1 to a grinding tube, quickly pressing for 1-2 times, and standing on ice for 3-5 min to prepare a tissue fluid;

Preferably, the lysis solution of steps S1 and S2 comprises sucrose with a molar mass of 250mM, Tris-HCl with a molar mass of 50mM, and molarCaCl with a mass of 50mM₂MgCl with a molar mass of 50mM₂DTT with the molar mass of 1mM, CA-630 with the mass percent of 0.1%, RNase Inhibitor with the unit of enzyme activity of 0.4U/. mu.L, BSA with the mass concentration of 0.04% and 1 × protease Inhibitor.

Preferably, the resuspension buffer described in steps S4, S5, S6 and S7 is 1 XPBS containing 0.04% by mass of BSA, 0.35M of mannitol, and 0.2U/. mu.L of RNase Inhibitor.

Preferably, the centrifugation process in step S4 is centrifugation at 1600-1700 rpm for 8-12S.

Preferably, the centrifugation process in the step S5 is centrifugation for 4-6 min at a rotation speed of 2600-2700 rpm; the mass percent of the iodixanol is 55-65%, and the adding amount is 0.6-0.8 time of the total volume of the liquid; and the rotating speed during re-centrifugation is 10000-12000 rpm, and the centrifugation time is 15-20 min.

Preferably, the centrifugation process in step S6 is centrifugation for 5-8 min at 2600-2700 rpm.

Preferably, the centrifugation process in the step S7 is centrifugation for 4-7 min at a rotation speed of 2600-2700 rpm; the sterilizing liquid comprises the following components in parts by weight: 5-10 parts of honeysuckle powder, 3-5 parts of allicin, 2-3 parts of fructus forsythiae extract and 50-60 parts of deionized water.

The invention also provides an application of the preparation method in constructing a single-cell transcriptome sequencing library.

Preferably, the cell suspension prepared using the method is placed on ice and the pool is built in 30 min.

In the invention, after the tissue is crushed, the cell is cracked by using a self-made lysate, and the cell nucleus is left, wherein the lysate contains an RNase Inhibitor, which can inhibit the activity of the RNase and prevent RNA degradation, and the RNase Inhibitor is matched with other components to ensure the integrity of the cell nucleus. Meanwhile, in the preparation method provided by the invention, after the cell sediment is prepared, the cell sediment is cleaned by using the sterilizing liquid, so that the possible bacterial and fungal pollution in the suspension preparation process is eliminated.

In addition, the inventors are trying toIn the test process, it is found that the lysis solution prepared by all tissues according to the following method can obtain good effect: 4070. mu.L sucrose + 600. mu.L Tris-HCl + 120. mu.L CaCl₂+180μL MgCl₂+ 5. mu.L DTT + 5. mu.L CA-630+ 5. mu.L RNase Inhibitor + 10. mu.L BSA + 5. mu.L 1 × protease Inhibitor; the resuspension buffer is used for preparing 5mL of 1 XPBS together, wherein the 1 XPBS also comprises BSA with the final concentration of 0.04 percent by mass, mannitol with the final concentration of 0.35M and RNase Inhibitor with the final enzyme activity of 0.2U/. mu.L; the BSA and mannitol were mixed with 1 XPBS, contained within 5 mL; when the cell nucleus sediment is diluted by 1 XPBS buffer solution, precooled 1 XPBS is added according to the quantity of the sediment, and the cell nucleus concentration is ensured to be 800-1500/mu L.

Compared with the prior art, the preparation method of the animal cell nucleus suspension provided by the invention has the following advantages:

(1) the preparation method provided by the invention realizes the preparation of the animal cell nucleus suspension for the first time;

(2) according to the preparation method provided by the invention, the cell lysis is carried out by adopting the self-made lysis solution, so that the RNA degradation is effectively prevented, and the integrity of cell nucleus is ensured;

(3) the preparation method provided by the invention also cleans the cell sediment by using the sterilizing liquid, thereby further ensuring that the cell suspension is not polluted by other bacteria.

Drawings

FIG. 1 is a graph showing the results of nuclear concentration measurements using a microscope counter plate;

FIG. 2 is a graph showing the results of the concentration of cell nuclei after trypan blue staining.

Detailed Description

The present invention is further explained with reference to the following specific examples, but it should be noted that the following examples are only illustrative of the present invention and should not be construed as limiting the present invention, and all technical solutions similar or equivalent to the present invention are within the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are commercially available products.

The honeysuckle powder can be purchased from Xian Huiban bioengineering limited company; the allicin is available from Shanghai Ji to Biochemical technology, Inc., and the fructus forsythiae extract is available from Shandong Nuphar Biotech, Inc.; the RNase Inhibitor is available from Nanjing Novozam Biotech, Inc.; the 1 × protease inhibitor is available from Guangzhou Hongkang Biotechnology, Inc.; the animal tissues selected in the examples of the invention are all liver tissues of mice, and the addition amount is 50 mg.

EXAMPLE 1 preparation of a suspension of animal cell nuclei

The preparation method of the animal cell nucleus suspension comprises the following steps:

s1, collecting 50mg of target tissue, adding 450 μ L of lysate, standing on ice for 4min, and cutting into 1mm³Size fraction to obtain tissue fraction containing lysate; the lysis solution comprises sucrose with the molar mass of 250mM, Tris-HCl with the molar mass of 50mM and CaCl with the molar mass of 50mM₂MgCl with a molar mass of 50mM₂DTT with the molar mass of 1mM, CA-630 with the mass percent of 0.1%, RNase Inhibitor with the unit of enzyme activity of 0.4U/. mu.L, BSA with the mass concentration of 0.04% and 1 × protease Inhibitor;

s2, transferring the tissue fragments containing the lysate prepared in the step S1 to a grinding tube, quickly pressing for 1 time, and standing on ice for 3min to prepare a tissue fluid;

s3, pressing the tissue fluid prepared in the step S2 for 8 times, standing on ice for 2min, pressing again for 14 times, and quickly transferring to a new centrifugal tube to prepare a homogenate mixed solution;

s4, adding 450 mu L of resuspension Buffer into the homogenate mixed liquor prepared in the step S3, reversing the mixture back and forth for 5 times, mixing the mixture evenly, then centrifuging the mixture for 8S at the rotating speed of 1600rpm, and transferring the supernatant into a 15mL centrifuge tube to prepare new homogenate; the resuspension Buffer contains 0.04 percent of BSA (bovine serum albumin), 0.35M of mannitol and 1 XPBS (phosphate buffered saline) of RNase Inhibitor with the activity of 0.2U/. mu.L by mass percentage;

s5, centrifuging the new homogenate obtained in the step S4 at 2600rpm for 4min, removing supernatant, adding 1mL of the resuspension buffer to fully resuspend the precipitate, repeating the steps for 2 times continuously, then adding iodixanol with the mass percent of 55% into the homogenate, wherein the addition is 0.6 time of the total volume, blowing and uniformly mixing the mixture, adding 700 mu L of the resuspension buffer into the mixture, centrifuging the mixture again at 10000rpm for 15min, removing impurities and supernatant by using a pipette gun, and keeping lower-layer liquid;

s6, adding 1mL of resuspension buffer into the lower layer liquid in the step S5, gently blowing, sucking and mixing uniformly, then centrifuging at 2600rpm for 5min, removing the supernatant, finally leaving 180 mu L in a centrifuge tube, adding 450 mu L of resuspension buffer into the centrifuge tube, and gently blowing, sucking and resuspending to prepare cell suspension;

s7, filtering the cell suspension prepared in the step S6 by using a cell sieve with the size of 40 microns, adding 450 microns of heavy suspension buffer into the cell suspension, centrifuging the cell suspension for 4min at the rotating speed of 2600rpm, collecting precipitates, then washing the precipitates for 2 times by using a sterilizing solution, centrifuging the precipitates, removing supernate, adding precooled 1 XPBS into the precipitates, and detecting the concentration of the precipitates to obtain the cell suspension; the sterilizing liquid comprises the following components in parts by weight: 5 parts of honeysuckle powder, 3 parts of allicin, 2 parts of fructus forsythiae extract and 50 parts of deionized water.

EXAMPLE 2 preparation of a suspension of animal cell nuclei

The preparation method of the animal cell nucleus suspension comprises the following steps:

s1, taking 50mg of target tissue, adding 550 mu L of lysis solution, standing on ice for 6min, and cutting the tissue into 3mm³Size fraction to obtain tissue fraction containing lysate; the lysis solution comprises sucrose with the molar mass of 250mM, Tris-HCl with the molar mass of 50mM and CaCl with the molar mass of 50mM₂MgCl with a molar mass of 50mM₂DTT with the molar mass of 1mM, CA-630 with the mass percent of 0.1%, RNase Inhibitor with the unit of enzyme activity of 0.4U/. mu.L, BSA with the mass concentration of 0.04% and 1 × protease Inhibitor;

s2, transferring the tissue fragments containing the lysate prepared in the step S1 to a grinding tube, quickly pressing for 2 times, and standing on ice for 5min to prepare a tissue fluid;

s3, pressing the tissue fluid prepared in the step S2 for 12 times, standing on ice for 3min, pressing again for 16 times, and quickly transferring to a new centrifugal tube to prepare a homogenate mixed solution;

s4, adding 550 mu L of resuspension Buffer into the homogenate mixed liquor prepared in the step S3, reversing the mixture back and forth for 8 times, mixing the mixture evenly, then centrifuging the mixture for 12S at the rotating speed of 1700rpm, and transferring the supernatant into a 15mL centrifuge tube to prepare new homogenate; the resuspension Buffer contains 0.04 percent of BSA (bovine serum albumin), 0.35M of mannitol and 1 XPBS (phosphate buffered saline) of RNase Inhibitor with the activity of 0.2U/. mu.L by mass percentage;

s5, centrifuging the new homogenate prepared in the step S4 at 2700rpm for 6min, removing supernatant, adding 2mL of the resuspension buffer to fully resuspend and precipitate, repeating for 2 times continuously, adding 65% by mass of iodixanol into the homogenate, wherein the addition amount is 0.8 times of the total volume, blowing and uniformly mixing the mixture, adding 700 mu L of the resuspension buffer into the mixture, centrifuging at 12000rpm for 20min again, removing impurities and supernatant by using a pipette gun, and keeping lower-layer liquid;

s6, adding 2mL of the heavy suspension buffer into the lower layer liquid in the step S5, carrying out soft blowing and sucking, mixing uniformly, then centrifuging at 2700rpm for 8min, removing the supernatant, finally leaving 220 mu L in a centrifuge tube, adding 550 mu L of the heavy suspension buffer into the centrifuge tube, and carrying out soft blowing and sucking for heavy suspension to prepare cell suspension;

s7, filtering the cell suspension prepared in the step S6 by using a cell sieve with the size of 40 microns, adding 550 mu L of heavy suspension buffer into the cell suspension, centrifuging the cell suspension for 7min at the rotating speed of 2700rpm, collecting precipitates, then cleaning the precipitates for 2 times by using a sterilizing solution, centrifuging the precipitates, removing supernate, adding precooled 1 XPBS into the precipitates, and detecting the concentration of the precipitates to obtain the cell suspension; the sterilizing liquid comprises the following components in parts by weight: 10 parts of honeysuckle powder, 5 parts of allicin, 3 parts of fructus forsythiae extract and 60 parts of deionized water.

EXAMPLE 3 preparation of a suspension of animal cell nuclei

The preparation method of the animal cell nucleus suspension comprises the following steps:

s1, collecting 50mg of target tissue, adding 500 μ L of lysis solution, standing on ice for 5min, and cutting into 2mm³Size fraction to obtain tissue fraction containing lysate; the lysis solution comprises cane with the molar mass of 250mMSugar, Tris-HCl 50mM in molar mass, CaCl 50mM in molar mass₂MgCl with a molar mass of 50mM₂DTT with the molar mass of 1mM, CA-630 with the mass percent of 0.1%, RNase Inhibitor with the unit of enzyme activity of 0.4U/. mu.L, BSA with the mass concentration of 0.04% and 1 × protease Inhibitor;

s2, transferring the tissue fragments containing the lysate prepared in the step S1 to a grinding tube, quickly pressing for 2 times, and standing on ice for 4min to prepare a tissue fluid;

s3, pressing the tissue fluid prepared in the step S2 for 10 times, standing on ice for 2.5min, pressing again for 15 times, and quickly transferring to a new centrifugal tube to prepare a homogenate mixed solution;

s4, adding 500 mu L of resuspension Buffer into the homogenate mixed liquor prepared in the step S3, reversing the mixture back and forth for 6 times, mixing the mixture evenly, then centrifuging the mixture for 10S at the rotating speed of 1650rpm, and transferring the supernatant into a 15mL centrifuge tube to prepare new homogenate; the resuspension Buffer contains 0.04 percent of BSA (bovine serum albumin), 0.35M of mannitol and 1 XPBS (phosphate buffered saline) of RNase Inhibitor with the activity of 0.2U/. mu.L by mass percentage;

s5, centrifuging the new homogenate obtained in the step S4 at 2650rpm for 5min, removing supernatant, adding 1.5mL of the resuspension buffer to fully resuspend and precipitate, continuously repeating for 2 times, then adding 60% by mass of iodixanol into the homogenate, wherein the addition amount is 0.7 times of the total volume, blowing and uniformly mixing the mixture, adding 700 mu L of the resuspension buffer into the mixture, centrifuging at 10658rpm again for 18min, removing impurities and supernatant by using a pipette gun, and keeping lower-layer liquid;

s6, adding 1.5mL of resuspension buffer into the lower layer liquid in the step S5, gently blowing, sucking and mixing uniformly, then centrifuging for 7min at the rotating speed of 2650rpm, removing the supernatant, finally leaving 200 mu L of resuspension buffer in a centrifuge tube, adding 500 mu L of resuspension buffer into the centrifuge tube, gently blowing, sucking and resuspending to prepare cell suspension;

s7, filtering the cell suspension prepared in the step S6 by using a cell sieve of 40 mu m, adding 500 mu L of heavy suspension buffer into the cell suspension, centrifuging the cell suspension for 5min at the rotating speed of 2650rpm, collecting precipitates, then cleaning the precipitates for 2 times by using a sterilizing solution, centrifuging the precipitates, removing supernate, adding precooled 1 XPBS into the precipitates, and detecting the concentration of the precipitates to obtain the cell suspension; the sterilizing liquid comprises the following components in parts by weight: 8 parts of honeysuckle powder, 4 parts of allicin, 2.5 parts of forsythia extract and 55 parts of double-purified water.

Comparative example 1 preparation of a suspension of animal cell nuclei