阅读说明：本技术 一种单细胞裂解液及其应用 (Single cell lysis solution and application thereof ) 是由 訾晓渊 雷佳 于 2021-02-22 设计创作，主要内容包括：本发明属于生物技术领域,具体涉及一种单细胞裂解液及其应用。本发明所提供的单细胞裂解液可以在常温状态下保持线粒体完整的同时快速裂解细胞,耗时短,避免裂解温度过高、耗时长导致的RNA降解与污染。其单细胞裂解液组份均为常用试剂,配制简单、方便、成本低。经验证,本发明所提供的单细胞裂解液在常温下仅需1-5min便可以实现单细胞的裂解,高效简捷,且裂解强度适中,裂解后线粒体依然完好；显著提高单细胞转录组测序成功率,具有较高的商业应用价值。(The invention belongs to the technical field of biology, and particularly relates to a single cell lysate and application thereof. The single cell lysate provided by the invention can rapidly lyse cells while keeping the integrity of mitochondria at normal temperature, has short time consumption, and avoids RNA degradation and pollution caused by overhigh lysis temperature and long time consumption. The components of the unicellular lysis solution are common reagents, and the preparation is simple and convenient, and the cost is low. The single cell lysis solution provided by the invention can realize the lysis of single cells only within 1-5min at normal temperature, is efficient and simple, has moderate lysis strength, and is still intact mitochondria after lysis; obviously improves the sequencing success rate of the single cell transcriptome and has higher commercial application value.)

1. A single cell lysate, which comprises 0.02-2% by mass of Triton X100, 0.05-0.5M of Tris-HCl, 0.1-1M of LiCl, 5-10 mM of DTT, and 10-20 mM of EDTA.

2. The lysate of claim 1, wherein the lysate of single cells comprises Triton X100 at a mass fraction of 0.02-1%, Tris-HCl at a final concentration of 0.05-0.2M, LiCl at a final concentration of 0.1-0.5M, DTT at a final concentration of 5-8 mM, and EDTA at a final concentration of 10-14 mM.

3. The lysate of claim 1, wherein the lysate of single cells comprises Triton X100 at a mass fraction of 0.02-1%, Tris-HCl at a final concentration of 0.05-0.1M, LiCl at a final concentration of 0.1-0.2M, DTT at a final concentration of 5mM, and EDTA at a final concentration of 10 mM.

4. The lysate of claim 1, wherein the single cell lysate comprises Triton X100 at a mass fraction of 0.02%, Tris-HCl at a final concentration of 0.05M, LiCl at a final concentration of 0.1M, DTT at a final concentration of 5mM, and EDTA at a final concentration of 10 mM.

5. A method for extracting single-cell nucleic acid in single-cell transcriptome sequencing by using the single-cell lysate of any one of claims 1 to 4.

6. The method of claim 5, wherein the sample is separated on the microfluidic chip to obtain single cells, and then mixed with the single cell lysate, and the single cell lysate is processed for 1-5min to obtain the nucleic acid contained in the sample after separation.

7. Use of a single cell lysate according to any of claims 1 to 4 for the extraction of nucleic acids from whole blood or cultured cells.

8. A kit for extracting nucleic acid from a cell, comprising the single cell lysate of any one of claims 1 to 4.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a single cell lysate and application thereof.

Background

Single cell transcriptome sequencing (scRNA-seq) is a new technology which is emerging in recent years, can obtain a whole transcriptome expression profile from a single cell level, and can perform high-throughput sequencing after amplification, so that the gene expression level in the single cell can be efficiently detected, and the method has important application value in the fields of development and differentiation of stem cells, diagnosis and treatment of tumors, design of targeted drugs and the like. The single cell sequencing process mainly comprises single cell extraction, cell lysis, mRNA reverse transcription, cDNA amplification, library construction, sequencing and data analysis. In recent years, a microfluidic system is mainly adopted to separate single cells from a cell suspension, and after the single cells are obtained through microfluidic separation, how to rapidly and efficiently crack the single cells to release mRNA in cytoplasm is very critical to the subsequent construction of an NGS library.

At present, the methods commonly used for lysing single cells mainly include freeze-thaw denaturation, alkaline lysis, proteinase K lysis, and the like. In the actual operation process, the cracking methods have defects of different degrees, such as complex operation, long time consumption, easy RNA degradation caused by overhigh temperature, overlarge cracking strength, breakage of cell nucleus and mitochondria during cell breakage and the like, and the accuracy of the subsequent sequencing result is seriously influenced.

Disclosure of Invention

In view of the above, the present invention provides a single cell lysate and applications thereof, aiming at the defects in the prior art. The cell lysate provided by the invention solves the problem that the cell nucleus and the mitochondria are simultaneously broken when the existing single cell is cracked.

In order to solve the technical problems, the invention is realized by the following technical scheme:

in one aspect of the invention, a single cell lysate is provided, wherein the lysate comprises 0.02-2% by mass of Triton X100, 0.05-0.5M of Tris-HCl, 0.1-1M of LiCl, 5-10 mM of DTT, and 10-20 mM of EDTA.

Preferably, in an embodiment of the present invention, the single cell lysate comprises Triton X100 at a mass fraction of 0.02 to 1%, Tris-HCl at a final concentration of 0.05 to 0.2M, LiCl at a final concentration of 0.1 to 0.5M, DTT at a final concentration of 5 to 8mM, and EDTA at a final concentration of 10 to 14 mM.

More preferably, in one embodiment of the present invention, the single cell lysate comprises Triton X100 at a mass fraction of 0.02 to 1%, Tris-HCl at a final concentration of 0.05 to 0.1M, LiCl at a final concentration of 0.1 to 0.2M, DTT at a final concentration of 5mM, and EDTA at a final concentration of 10 mM.

More preferably, in one embodiment of the present invention, the single-cell lysate contains 0.02% by mass of Triton X100, 0.05M final Tris-HCl, 0.1M final LiCl, 5mM final DTT, and 10mM final EDTA.

In another aspect of the invention, a method for single cell nucleic acid extraction in single cell transcriptome sequencing is provided. The method is carried out using a single cell lysate as described in any of the above.

In the method, a sample is separated on a microfluidic chip to obtain single cells, then the single cells are mixed with the single cell lysate, the mixture is processed for 1-5min, and nucleic acid contained in the sample is obtained after separation.

In another aspect of the invention, a kit for extracting nucleic acid is provided, and the kit comprises the single cell lysate.

In another aspect of the invention, the single cell lysate is provided for extracting nucleic acid from whole blood or cells in single cell transcriptome sequencing.

Compared with the prior art, the invention has the beneficial effects that:

the single cell lysate provided by the invention can rapidly lyse cells while keeping the integrity of mitochondria at normal temperature, has short time consumption, and avoids RNA degradation and pollution caused by overhigh lysis temperature and long time consumption. The components of the single cell lysis solution provided by the invention are common reagents, and the single cell lysis solution is simple and convenient to prepare and low in cost. Verification proves that the lysis solution provided by the invention can realize the lysis of single cells only in 1-5min at normal temperature, is efficient and simple, has moderate lysis strength, and the mitochondria are still intact after lysis; obviously improves the sequencing success rate of the single cell transcriptome and has higher commercial application value.

Drawings

FIG. 1 is a microscope image of a lysed microfluidic chip before cell lysis;

FIG. 2 is a microscope image of lysed cells in a microfluidic chip after lysis.

Detailed Description

The invention discloses a single cell lysate and application thereof, and a person skilled in the art can appropriately improve process parameters by referring to the content. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. The experimental procedures, in which specific conditions are not specified in the examples below, are generally carried out according to conventional conditions or according to conditions recommended by the manufacturers. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. In addition, any methods and materials similar or equivalent to those described herein can be used in the methods of the present invention. The preferred embodiments and materials described herein are intended to be exemplary only.

Materials: tris (hydroxymethyl) aminomethane (Tris-HCl), lithium chloride (Licl), polyethylene glycol octylphenyl ether (Triton X100), Dithiothreitol (DTT), ethylenediaminetetraacetic acid (EDTA) were purchased from Biotechnology engineering (Shanghai) Inc.; the microfluidic chip is from New Geyuan Nanjing Biotechnology Ltd.

Example 1: preparation of single cell lysate

The components of the single cell lysate can adopt the following component proportion of the single cell lysate in the embodiment when in specific application: preparing 1mL of single cell lysate, sucking 2 mol/L of Tris-HCl 50uL into a centrifuge tube by using a pipette gun, adding 5mol/L of Licl 100uL, adding 20% of Triton X1005 uL, adding 1mol/L of EDTA 20 uL, adding 100mmol/LDTT 50uL, blowing, sucking, uniformly mixing, adding 775 uL of sterile water to supplement to 1mL, and mixing for later use.

Example 2: preparation of single cell lysate

The components of the single cell lysate can adopt the following component proportion of the single cell lysate in the embodiment when in specific application: preparing 1mL of single cell lysate, sucking 2 mol/L Tris-HCl 50uL into a centrifuge tube by using a pipette gun, adding 5mol/L Licl 100uL, adding 20% Triton X1005 uL, adding 1mol/L EDTA 20 uL, adding 100mmol/LDTT 50uL, adding 775 uL sterile water, and mixing for later use.

Example 3: lysis of 293T cells

The 293T cells have high growth speed, the doubling time is generally not more than 1 day, the primary cells are recovered and then transferred to one generation for subsequent tests, single cells are obtained by separating 293T cell suspension with good growth state by utilizing a microfluidic chip in the embodiment, 100uL of the single cell lysate prepared in the embodiment 1 is added into the microfluidic chip, the cell lysis conditions before and after addition are observed under a microscope, the lysis time is recorded, and the lysis process is recorded by photographing.

FIG. 1 is a microscope image of a lysed microfluidic chip before cell lysis; FIG. 2 is a microscope image of lysed cells in a microfluidic chip after lysis; as can be seen from FIGS. 1 and 2, before the addition of the single cell lysate, 293T cells falling into the microfluidic chip have clear outlines and clearly visible nuclei. After the single cell lysate is added, the cells are completely lysed, the mitochondria are hardly lysed, and the time for completely lysing the cells is not more than 2 min.

Example 4: lysis of PBMC (peripheral blood mononuclear cells)

Taking normal human peripheral blood, preparing PBMC suspension of peripheral blood mononuclear cells, dyeing the PBMC suspension by using a cell nucleus dyeing agent, separating the dyed PBMC suspension by using a microfluidic chip to obtain single cells, adding 100uL of the single cell lysate prepared in the embodiment 2 into the microfluidic chip, observing the lysis condition of the peripheral blood mononuclear cells PBMC under a microscope, and recording the lysis time and the lysis process. The observation shows that the peripheral blood mononuclear cells are completely lysed within 3min after the single cell lysate prepared in the example 2 is added.

Example 5: effect of Single cell lysate on mitochondria

293T cells were routinely cultured and randomly divided into 4 groups of 3 replicates each, experimental group A (lysate from example 1), experimental group B (lysate from example 2), comparative group C (freeze-thaw lysis) and comparative group D (alkaline lysis). The culture solution was poured out of each group, washed 3 times with PBS buffer solution, and drained. Respectively adding corresponding single cell lysate into the experimental group A and the experimental group B, wherein the freeze-thaw lysis method specifically comprises the following steps: freezing at-20 deg.C, thawing, adding paraffin oil, and heat-denaturing at 95 deg.C for 35min to lyse the cells. The cracking liquid in the alkali cracking method comprises the following specific components: 1mol/L Tris-HCl, 0.5mol/L Licl, 1% SDS; the lysis time was 15 s. The lysis time and status of each group are shown in table 1.

TABLE 1 comparison of the degree of cleavage and the state of each group

Method	Cracking time	Intensity of cracking	Splitting mitochondria
				Experimental group A	1-5min	Is moderate	Small amount of
Experimental group B	1-5min	Is moderate	Small amount of
				Comparative group C	35min-14h	Weak (weak)	Small amount of
Comparison group D	1-15s	High strength	A large number of

As can be seen from Table 1, compared with the currently common freeze-thaw lysis method, the single cell lysate provided by the invention can lyse single cells within a shorter time (1-5 min), and the lysis intensity is moderate; although the alkaline lysis method can realize rapid cell lysis in 1-15s, mitochondria are also greatly lysed while single cells are lysed, which affects the accuracy of subsequent experiments. The integrity of mitochondria in the cell after the single cell lysate is cracked is obviously higher than that of the traditional alkali cracking method, the cracking intensity is moderate, the cell is cracked completely within 5 minutes, and compared with the existing cracking method, the single cell lysate has obvious advantages of time or cracking intensity.

Furthermore, the contents of mitochondria after cracking in each group of the above experiment are counted, and table 2 is a comparison table of the statistics of the contents of mitochondria in each group.

TABLE 2 mitochondrial content statistics comparison table

Rate of mitosis	Experimental group A	Experimental group B	Ratio pair group C	Ratio pair group D
					>5%	14.781491	15.2365931	48.344371	91.366655
>10%	1.7994859	1.787592	22.102649	50.716533
					>15%	0.8997429	0.3154574	12.417219	13.946173
>20%	0.5141388	0.3154574	5.380795	3.739951

As can be seen from Table 2, the mitochondria in the cell are still intact after the lysis of the single cell lysate provided by the present application, and the single cell lysate provided by the present application can be expected to significantly improve the sequencing success rate of the single cell transcriptome, and has obvious technical advantages.

The above description is only a preferred embodiment of the present invention, and is not intended to limit the scope of the present invention. It should be noted that, for those skilled in the art, without departing from the principle of the present invention, several improvements and modifications can be made, and these improvements and modifications should also be construed as the protection scope of the present invention.