CN115896241A - Preparation method of multiple single-cell miRNA sequencing library based on digital microfluidic chip - Google Patents
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Abstract
The invention discloses a preparation method of a plurality of single cell miRNA sequencing libraries based on a digital micro-fluidic chip, wherein the digital micro-fluidic chip is provided with at least one liquid storage area, at least one liquid drop generating channel, a single cell capturing area and a plurality of library reaction areas which are sequentially communicated and provided with super-hydrophobic surfaces, the at least one liquid storage area, the at least one liquid drop generating channel, the single cell capturing area and the plurality of library reaction areas are respectively provided with electrodes which are electrically connected with driving electrodes of the digital micro-fluidic chip, the single cell capturing area is provided with a plurality of hydrophilic parts which are uniformly distributed, and each hydrophilic part is correspondingly communicated with one library reaction area. The invention can realize the high-efficiency, automatic and selective simultaneous separation of a plurality of single cells, can carry out highly-parallelized miRNA library preparation, and can realize the miRNA detection with high sensitivity, low pollution and low cost.
Description
Technical Field
The invention belongs to the technical field of digital microfluidic chips, and particularly relates to a preparation method of a plurality of single-cell miRNA sequencing libraries based on a digital microfluidic chip.
Background
The cell is the basic unit of life activity, and can fully reveal the activity and regulation rule of a complex life system by researching the life processes of organism development, heredity, diseases and the like at the cell level. With the progress of cell research, researchers have found that even cells exhibiting the same phenotype or similar function may have differences, i.e., cell heterogeneity. Heterogeneity among individual cells plays a critical role in the development of living systems, and is a source of diversity in tissues, organs, and systems. However, most of the traditional cell researches are based on population level sampling analysis, so that the difference between single cells is covered, and the unique life behavior information of the single cells cannot be obtained. Therefore, the study on the heterogeneity of single cells is very important, and the development track of a living system, a disease worsening mechanism, an organism regulation rule and the like can be disclosed.
The heterogeneity among single cells is caused by the difference of genetic materials and gene expression conditions, and the single cell sequencing technology is a technology for comprehensively and finely analyzing the genome, transcriptome, epigenetic modification on the genome or transcriptome and chromatin structure of the single cell by applying a high-throughput sequencing technology. Through single cell sequencing, researchers can obtain information at multiple molecular layers and reveal the source of heterogeneity among single cells through interaction and regulation processes during information transmission among different molecules.
In the transcriptome, small RNA is a non-coding RNA molecule with the length shorter than 300nt, and can play an important regulation role in the processes of transcription, processing and translation of RNA. Among the small RNAs, miRNA (microRNA) is a small RNA with the highest expression heterogeneity among single cells, and can be specifically hybridized with target mRNA to cause the translation inhibition or degradation of the target mRNA, thereby regulating the synthesis of downstream protein and the display of cell functions, and playing a vital role in the life processes of cell development, activity, diseases and the like. Currently, miRNA has become a disease marker of many cancers, and therefore, sequencing analysis of miRNA is crucial.
However, the development of the single cell miRNA sequencing technology is not completely mature at present, and the currently reported single cell miRNA sequencing methods are all processes for completing the construction of the whole sequencing library in a reaction system based on a centrifuge tube, and single cells need to be manually and sequentially selected into the centrifuge tube one by one under microscopic imaging, so that the operation is complicated, the time consumption is long, and pollution is easily introduced. In addition, the reaction system based on the centrifuge tube requires a larger reaction volume, which may result in limited sensitivity, accuracy and reproducibility of the biomolecule reaction, and a larger reagent consumption, increasing the preparation cost of the sequencing library.
The micro-fluidic technology is a device which is used for preparing a chip containing a micro-channel or a micro-cavity by a micro-nano processing means, can process fluid with extremely small volume (can be as low as nanoliter to attoliter) and is configured with functionalization. The microfluidic chip can integrate and miniaturize complex functions of a traditional laboratory onto a single chip so as to solve the problem which is difficult to solve in the traditional experiment or improve the reaction efficiency, and is a new technology combining the subjects of chemistry, microelectronics, materials, biomedicine, fluidics and the like. The development of the microfluidic technology provides a new approach for preparing a single-cell sequencing library, a plurality of new single-cell mRNA sequencing technologies based on the traditional microfluidic technology are developed at present, and the cell analysis flux, the gene detection sensitivity and the like can be remarkably improved. However, miRNA cannot be specifically captured by a fixed sequence target like mRNA, and the sequencing library has a long process and involves multiple sample addition reactions, so that the current microfluidic platform (including a droplet microfluidic chip, an integrated microfluidic chip, and a microarray chip) commonly used for sequencing single-cell mRNA is difficult to be applied to preparation of a single-cell miRNA sequencing library, and the above techniques are difficult to realize selective cell capture, are difficult to avoid for single cells or cell fragments with poor states, and are easy to cause certain resource waste. Therefore, a single-cell miRNA sequencing platform which can realize efficient, simple and selective simultaneous separation of multiple single cells, integrate a multistep miRNA sequencing library preparation process, and improve the detection capability of single-cell mirnas is lacking at present.
Disclosure of Invention
The invention aims to overcome the defects of the prior art and provides a preparation method of a plurality of single-cell miRNA sequencing libraries based on a digital microfluidic chip.
The technical scheme of the invention is as follows:
a preparation method of a plurality of single-cell miRNA sequencing libraries based on a digital micro-fluidic chip is disclosed, the digital micro-fluidic chip is provided with at least one liquid storage area, at least one liquid drop generation channel, a single-cell capture area and a plurality of library reaction areas which are sequentially communicated and provided with super-hydrophobization surfaces, the at least one liquid storage area, the at least one liquid drop generation channel, the single-cell capture area and the plurality of library reaction areas are respectively provided with electrodes which are electrically connected with driving electrodes of the digital micro-fluidic chip, the single-cell capture area is provided with a plurality of hydrophilic parts which are uniformly distributed, and each hydrophilic part is correspondingly communicated with one library reaction area;
the preparation method comprises the following steps:
(1) Delivering the cell suspension to the at least one reservoir region under conditions in which the electrodes of the at least one reservoir region are energized;
(2) Electrically switching the electrodes of the at least one droplet generation channel to separate droplets of the cell suspension from the cell suspension in the at least one reservoir region;
(3) Electrically controlling the on-off of at least one droplet generation channel and the electrodes of the single cell capturing area to enable the droplets of the cell suspension to be displaced into the hydrophilic part of the single cell capturing area, enabling one single cell in the droplets of the cell suspension to be opposite to the hydrophilic part, then powering off and standing to enable the single cell to naturally settle to the bottom of the hydrophilic part under the action of gravity, and then removing the droplets of the cell suspension to enable the single cell to remain at the bottom of the hydrophilic part;
(4) De-energizing the electrodes of at least one of the reservoir zones and washing the remaining cell suspension therein with sterile water;
(5) Carrying out on-off control on electrodes of at least one liquid storage area, at least one liquid drop generation channel and a single cell capture area, conveying cell lysate to a hydrophilic part with single cells settled at the bottom from the at least one liquid storage area, carrying out cell lysis reaction with the single cells, heating by a heating device to fully release miRNA from the cell lysate, carrying out on-off control on the electrodes of the single cell capture area and a plurality of library reaction areas, and conveying materials positioned in the hydrophilic part to the corresponding library reaction areas;
(6) According to the step (5), repeatedly carrying out on-off control on electrodes of at least one liquid storage area, at least one liquid drop generation channel, a single cell capture area and a plurality of library reaction areas, sequentially moving a 3 'joint connection reagent, an excess 3' joint digestion reagent, a5 'joint connection reagent, a first reverse transcription pre-reaction reagent, a second reverse transcription pre-reaction reagent and a reverse transcription reagent from the at least one liquid storage area to the corresponding library reaction areas, and sequentially completing a 3' joint connection reaction, an excess 3 'joint digestion reaction, a 5' joint connection reaction, a first reverse transcription pre-reaction, a second reverse transcription pre-reaction and a reverse transcription reaction by heating through a heating device;
(7) Adding an amplification reagent into a material obtained after completion of reverse transcription reagents in a plurality of library reaction regions, and after fully mixing, carrying out a first amplification reaction by a gene amplification device;
(8) And (4) mixing the materials from different library reaction regions obtained in the step (7) with miRNA library construction amplification reaction reagents respectively, and carrying out a second amplification reaction through an amplification device to obtain miRNA sequencing libraries corresponding to different single cells.
After each round of adding different reagents is finished, the surface of the liquid storage area is cleaned by enzyme-free sterile water, so that cross contamination among the reagents is prevented.
In a preferred embodiment of the invention, the formulation of the cell lysate is:
in a preferred embodiment of the present invention, the formulation of the 3' linker connecting reagent is:
in a preferred embodiment of the invention, the formulation of the excess 3' linker digesting agent is:
in a preferred embodiment of the present invention, the formulation of the 5' linker linking reagent is:
in a preferred embodiment of the present invention, the formulation of the first reverse transcription pre-reaction reagent is:
the formula of the second reverse transcription pre-reaction reagent is as follows:
the formula of the reverse transcription reaction reagent is as follows:
in a preferred embodiment of the present invention, the formulation of the amplification reagent is:
in a preferred embodiment of the present invention, the formulation of the miRNA library-building amplification reaction reagent is:
in a preferred embodiment of the present invention, the hydrophilic portion is a circular groove having a surface that is hydrophilic and has a diameter of 90 to 110 μm, which is formed by a partial peeling technique.
In a preferred embodiment of the present invention, the number of the liquid storage regions is two, the number of the droplet generation channels is two, and the single-cell trapping region has a seven hydrophilic portion.
The invention has the beneficial effects that:
1. the invention can realize the high-efficiency, automatic and selective simultaneous separation of a plurality of single cells: the designed digital microfluidic chip is constructed with a plurality of hydrophilic parts, and can efficiently and rapidly complete the selective separation and capture of a plurality of single cells in a short time, so that the system ensures that the activity state of the captured single cells is ensured while the cell analysis throughput is improved, and the output quality of the single cell miRNA library is further ensured.
2. The invention can carry out highly parallelized miRNA library preparation: based on the uniformity generated by the digital microfluidic droplets and the capability of parallel control of discrete droplets, the steps of cracking of a plurality of single cells, double-end joint connection of miRNA, reverse transcription and amplification are simultaneously completed on a single digital microfluidic chip, the highly parallel flow reduces batch difference among single cell samples, improves the accuracy, stability and reproducibility of system output, and enables sequencing results to reflect the heterogeneity among the single cells.
3. The miRNA library construction method realizes the miRNA detection with high sensitivity, low pollution and low cost: the digital microfluidic chip reacts in a closed oil phase space, and the influence of external air pollution on a reaction system during reaction can be isolated. In addition, compared with the micro-upgraded reaction volume in a centrifugal tube, the micro-upgraded reaction volume in the digital microfluidic chip can greatly improve the effective reaction concentration of miRNA molecules, improve the miRNA detection capability, and simultaneously can effectively reduce the consumption of reagents, thereby reducing the reagent cost of the system.
Drawings
Fig. 1 is a schematic structural diagram of a digital microfluidic chip in an embodiment of the present invention.
FIG. 2 is a comparison of miRNA numbers measured at different sequencing depths for single-cell miRNA samples prepared by the centrifugal tube reaction system of the present invention and a traditional centrifugal tube reaction system in example 4 of the present invention.
FIG. 3 is a graph showing the correlation analysis of the miRNA expression level between the data set of 10 non-small cell lung cancer cells H2228 cells prepared by the method and the H2228 cell bulk sample prepared by the centrifugal tube in example 4 of the invention.
FIG. 4 is a schematic diagram showing the differential expression of 20H 2228 intercellular miRNAs measured by the present invention in example 4 of the present invention.
FIG. 5 is a diagram showing miRNAs differentially expressed among three non-small cell lung cancer cell lines A549, H1975 and H2228 measured by the present invention in example 4 of the present invention.
FIG. 6 is a schematic diagram of UMAP dimension reduction analysis performed according to the miRNA differential expression of three non-small cell lung cancer cell lines A549, H1975 and H2228 in example 4 of the present invention.
Detailed Description
The technical solution of the present invention will be further illustrated and described below with reference to the accompanying drawings by means of specific embodiments.
Example 1: cell line and culture conditions
The cell lines used in the present invention include: human non-small cell lung cancer cell lines H2228, H1975, A549. All cells were cultured and stored for a long period in the inventors' laboratory. The cell lines were all cultured in RPMI medium containing 10% FBS, 1% penicillin/streptomycin and 60mm dishes, and the cells were cultured at 37 ℃ with 5% CO 2 The incubator of (1). When the cells need to be passaged or collected for subsequent experiments, the upper layer culture medium of the cells is completely absorbed, the cells are washed once by 1 XPBS, trypsin is added for digestion for a corresponding time, the trypsin is completely absorbed after digestion is finished, 2mL of complete culture medium is added to stop digestion, and a part of the cells are taken out for passage or subsequent experiments after the cells are fully blown and beaten.
Example 2: digital microfluidic chip structure design
The digital microfluidic chip provided by the invention is provided with two liquid storage areas, two droplet generation channels, a single cell capture area and a seven library reaction area which are sequentially communicated and are provided with super-hydrophobic surfaces, wherein the liquid storage areas, the droplet generation channels, the single cell capture area and the library reaction area are all provided with electrodes which are electrically connected with driving electrodes of the digital microfluidic chip, the single cell capture area is provided with seven hydrophilic parts which are uniformly distributed, and each hydrophilic part is correspondingly communicated with one library reaction area;
as shown in fig. 1: the region 1 is a driving electrode access port, is 96 in number, and is respectively connected with the electrodes of the liquid storage region, the liquid drop generation channel, the single cell capture region and the library reaction region one by one; the area 2 is a liquid storage area, single cell suspension, cell lysate, a 3' joint connecting reagent, a 3' joint digesting reagent, a 5' joint connecting reagent, a reverse transcription reagent and an amplification reagent are all led into the digital microfluidic chip through the area, the liquid storage area comprises two liquid storage tank electrodes, and the size of the electrodes is 1.7354mm multiplied by 1.2mm; a droplet generation channel is arranged between the area 2 and the area 3 and comprises two droplet generation channels connected with 3 electrodes, and the size of the electrodes in the area is 0.6mm multiplied by 0.6mm; the region 3 is a single cell capture region, which comprises 7 hydrophilic parts corresponding to the reaction regions of the library one by one and is used for separating and capturing single cells, and the electrode size of the region is 0.8mm multiplied by 0.8mm; the large electrodes connected with the left part and the right part of the single cell capturing area are miRNA library reaction areas, the single cell capturing area is connected with the single cell capturing area through two, one or two electrodes with the size of 0.8mm multiplied by 0.8mm, and the electrode size of the library reaction area is 2.7mm multiplied by 1.8mm; the hydrophilic part is a circular groove with a diameter of 100 μm and a hydrophilic surface formed by local peeling
The manufacturing process of the upper plate and the lower plate of the chip is carried out according to Q.Y.Ruan, J.Yang, F.X.Zou, X.F.Chen, et al, anal Chem.2022, 94, 1108-1117. 1
Example 3: and (3) preparing a single-cell miRNA sequencing library based on the digital microfluidic chip.
This example was carried out using the cells of example 1 and the digital microfluidic chip of example 2, as follows:
(1) Moving the upper edge of the upper polar plate to the upper edge of a liquid storage area of the chip, pre-electrifying a liquid storage pool electrode, adding cell suspension to the edge of the liquid storage pool, and enabling liquid drops to automatically enter the center of the electrified electrode;
(2) Applying power-on and power-off control to the chip generation channel electrode according to a certain program through a computer-side liquid drop driving control program so as to generate a cell suspension liquid drop from the electrode of the liquid storage area, moving the liquid drop to the leftmost hydrophilic part by controlling the driving electrode, and adjusting the position of the liquid drop at the hydrophilic part;
(3) And (3) enabling single cells suspended in the liquid drops to face the hydrophilic part below, powering off and standing for 2min, enabling the cells to naturally settle to the bottom of the hydrophilic part under the action of gravity, dragging the liquid drops of the cell suspension at the moment, and leaving small liquid drops containing the single cells on the hydrophilic part. The same cell suspension liquid drop introduced into the chip can be used for sequentially capturing 7 hydrophilic part single cells;
(4) Cutting off the electrode in the liquid storage area, cleaning and removing residual cell suspension in the liquid storage area while cleaning the edge of the upper polar plate by 200 mu L of enzyme-free sterile water;
(5) Moving the upper edge of the upper polar plate to the upper edge of a liquid storage area at the left part of the chip, pre-electrifying the liquid storage area, adding cell lysate to the liquid storage area, sequentially generating 7 lysate liquid drops according to the step (2) and respectively moving the lysate liquid drops to each hydrophilic part, observing that the edge of the cells at the hydrophilic part becomes fuzzy after the cell lysate is introduced, and transferring cell lysate to a refrigerator at the temperature of-80 ℃ for storage;
TABLE 1 cell lysate formulation
(6) Placing the chip containing the cell lysate on a heating instrument for heating, so that miRNA is fully released from the cell lysate, after the heating is finished, moving the cell lysate liquid drops of the hydrophilic part to the corresponding hydrophilic part, and preparing for carrying out a subsequent reaction process;
(7) Moving the upper edge of the upper polar plate to the upper edge of a liquid storage area at the left part of the chip, pre-electrifying the liquid storage area, adding a 3' sequencing joint connecting reagent to the liquid storage area according to the table 2, applying on-off control on a chip generation channel electrode according to a certain program through a computer-side liquid drop driving control program, sequentially generating 7 liquid drops containing the 3' sequencing joint connecting reagent from the electrode of the liquid storage area, respectively moving the liquid drops to 7 hydrophilic parts, fully mixing the liquid drops with cell lysate, placing the liquid drops in a heating instrument to complete the 3' sequencing joint connecting reaction of miRNA, wherein the reaction conditions are heating at 30 ℃ for 6h, and cooling at 4 ℃ for 10h.
TABLE 2 3' linker ligation reaction reagent formulation for miRNA
(8) The addition of the excess 3 'linker digestion reagent was completed according to the sample addition procedure consistent with step (7), and the excess 3' linker digestion reagent formulation was set in a heating apparatus at 30 ℃ for 15min and 37 ℃ for 15min, as shown in Table 3.
TABLE 3 digestion reaction recipe for excess 3' linker of miRNA
(9) According to the sample adding step consistent with the step (7), the addition of the 5 'linker connecting reagent is completed, and the 5' linker connecting reaction reagent formula is shown in Table 4, and the mixture is placed in a heating apparatus to react for 1h at 37 ℃.
TABLE 4 5' linker ligation reaction reagent formulation for miRNA
(10) And (3) completing the addition of a first reverse transcription pre-reaction reagent according to the sample adding step consistent with the step (7), wherein the formula of the first reverse transcription pre-reaction reagent is shown in the table 5, and the first reverse transcription pre-reaction reagent is placed in a heating instrument to react for 5min at 65 ℃.
TABLE 5 first reverse transcription Pre-reaction reagent formulation
And (3) adding a second reverse transcription pre-reaction reagent according to the sample adding step consistent with the step (7), wherein the formula of the second reverse transcription pre-reaction reagent is shown in the table 6, and the second reverse transcription pre-reaction reagent is placed in a heating instrument to react for 30min at 42 ℃.
TABLE 6 second reverse transcription Pre-reaction reagent formulation
And (4) completing the addition of the reverse transcription reaction reagent according to the sample adding step consistent with the step (7), wherein the formula of the reverse transcription reaction reagent is shown in the table 7, and the reverse transcription reaction reagent is placed in a heating instrument to react for 2 hours at 42 ℃.
TABLE 7 Reversal reaction list reagent formula
(11) After the preparation of amplification reagents was completed according to Table 8, 0.3. Mu.L of each of the library reaction regions was directly added to the mixture, and after the droplets were sufficiently mixed, the chip was placed in a gene amplification apparatus to perform a first amplification reaction according to the procedure of Table 9.
TABLE 8 reverse transcription reagent formulations
TABLE 9 first amplification reaction procedure
(12) And taking the amplified liquid drops out of the chip, transferring the liquid drops into a centrifuge tube, adding miRNA library building amplification reaction reagents in the table 10 to enable the total volume to be 26 mu L, and carrying out amplification according to the amplification program in the table 11 to complete the library building reaction (second amplification reaction) of miRNA so as to obtain a library building product. The library products were purified using 1.2 × VAHTS DNA Clean Beads (Novozam) and then put on the machine for Illumina next generation sequencing.
TABLE 10miRNA library construction amplification reaction reagent formula
TABLE 11miRNA library construction and amplification procedure
Example 4: performance characterization of single cell miRNA detection analysis by using method of the invention
(1) Comparison of miRNA detection number with reaction system based on centrifugal tube
In order to compare the detection performances of the mirnas in two different reaction environments, namely a chip and a centrifuge tube, and verify the advantages of the invention, the miRNA library is constructed in the centrifuge tube by adopting a miRNA reaction process consistent with the chip. Wherein, picking single cells under a microscope and putting the single cells into a centrifuge tube to ensure that the volume of the single cells is 0.5 muL, adding 1.5 muL of cell lysate, and the formula and the heating procedure of the reaction reagent in the subsequent steps are consistent with those in the embodiment 3, wherein the volume of the reagent added into the centrifuge tube in the steps of 3' joint connection, 3' joint digestion, 5' joint connection, reverse transcription pre-reaction step I, reverse transcription pre-reaction step II and reverse transcription reaction is 2 muL, and the volume of the reagent added into the centrifuge tube in the amplification reaction of miRNA is 14 muL. After completion of the amplification reaction, 1. Mu.L of the amplification product was subjected to a pooling reaction, the total volume of which was 26. Mu.L, in accordance with example 3 (12).
The preparation of the centrifuge tube samples of 3 non-small cell lung cancer cells H2228 is completed according to the steps, the preparation of 3H 2228 cell chip samples is completed according to the embodiment 3, the number of miRNAs which can be measured in two reaction systems under different sequencing depths (0.25M, 0.5M, 1M, 2M and 5M) is compared, the result is shown in figure 2, it can be seen that the number of single-cell miRNAs detected by the chip samples under each sequencing depth is far higher than that of single-cell miRNAs detected by the centrifuge tube samples, and the invention proves that the detection capability of the single-cell miRNAs can be obviously improved by comparing the miRNA reaction system based on digital microfluidics with the traditional centrifuge tube reaction system.
(2) Verifying the accuracy of the miRNA information obtained by the invention
10H 2228 cell samples are prepared according to the method, and in addition, bulk samples of 500 cells are prepared, the process flow is consistent with the single cell sample preparation process flow based on a centrifuge tube reaction system, and only the difference is different from the input amount of the cells in the first step. After the sequencing data is obtained, the miRNA data of 10 cells are collected, the correlation between the data collection and the expression quantity of each miRNA in the bulk sample is analyzed, the result is shown in figure 3, the correlation coefficient is about 0.89, the correlation is good, and the accuracy of the single-cell miRNA information obtained based on the digital microfluidic reaction system is proved.
(3) The invention can reveal miRNA information of differential expression among single cells of the same cell line.
After 20H 2228 cell samples were prepared according to the method of the present invention and sequencing data were obtained, the standard deviations of the expression levels of each miRNA among 20 single cells were ranked according to the corresponding mean expression levels, and the results are shown in fig. 4, where each point represents one miRNA and the horizontal axis represents the miRNA expression level after normalization (log 2 (RPM + 0.25)). When the RPM corresponding to the miRNA is more than 100000, the miRNA is in ultrahigh expression level; when the corresponding RPM is less than 100000 and greater than 10000, the miRNA is at a high expression level; when the corresponding RPM is less than 10000 and greater than 100, the miRNA is at an intermediate expression level; when the corresponding RPM is less than 100, the miRNA is at a low expression level. 2 When the value interval of the point corresponding to the x axis in the figure is 0-6.4 by conversion according to the normalization processing mode,the miRNA with low expression level is the miRNA with medium expression at the point between 6.4 and 13 of the x-axis numerical interval, and the miRNA with high expression at the point between 13 and 16.4 of the x-axis numerical interval. As can be seen from the distribution of each point in the graph, the expression level of most of the miRNAs which are more differentially expressed among the single cells is lower, the expression difference of the high-expression miRNAs among the single cells is smaller, and the analysis result is consistent with the situation reported in the literature. 3
In addition, the figure also reveals that the most differentially expressed miRNAs between H2228 single cells are hsa-miR-148a-3p, hsa-miR-19b-3p and hsa-miR-101-3p, wherein the average expression level of hsa-miR-148a-3p and hsa-miR-19b-3p in the cells is low, and the expression level of hsa-miR-101-3p is medium; in addition, hsa-miR-103a-3p is miRNA with high expression quantity in H2228 cells and high degree of difference of cell-to-cell expression, but the degree of difference of cell-to-cell expression is not as high as the former; and the hsa-let-7a-5p is the miRNA with the highest average expression level and smaller expression difference in the H2228 cells. The data prove that the invention can fully reveal the miRNA expression difference among single cells of the same cell line and can fully research the heterogeneity among the single cells.
(4) The verification of the invention can reveal the difference of miRNA expression among different cell lines, and can distinguish cells according to the difference of miRNA expression.
After 10H 2228 cell samples, 10 a549 cell samples and 10H 1975 cell samples were prepared according to the method of example 3 of the present invention, and sequencing results were obtained, normalization processing and differential expression analysis were performed on miRNA expression levels of each cell line, and the results are shown in fig. 5, which proves that the present invention can sufficiently reveal mirnas differentially expressed between different cell lines. And then, UMAP dimension reduction analysis is carried out according to the miRNA condition of differential expression, the result is shown in figure 6, and the three cells can be obviously distinguished, so that the invention can be seen to well distinguish single cells of different cell types, and provide a new powerful analysis means for the future research of single cell miRNA.
The above description is only a preferred embodiment of the present invention, and therefore should not be taken as limiting the scope of the invention, which is defined by the appended claims.
Claims (10)
1. A preparation method of a plurality of single-cell miRNA sequencing libraries based on a digital microfluidic chip is characterized by comprising the following steps: the digital microfluidic chip is provided with at least one liquid storage area, at least one liquid drop generating channel, a single cell capturing area and a plurality of library reaction areas which are sequentially communicated and provided with super-hydrophobization surfaces, wherein the at least one liquid storage area, the at least one liquid drop generating channel, the single cell capturing area and the plurality of library reaction areas are respectively provided with an electrode which is electrically connected with a driving electrode of the digital microfluidic chip, the single cell capturing area is provided with a plurality of hydrophilic parts which are uniformly distributed, and each hydrophilic part is correspondingly communicated with one library reaction area;
the preparation method comprises the following steps:
(1) Delivering the cell suspension to the at least one fluid reservoir region under the condition that the electrodes of the at least one fluid reservoir region are energized;
(2) Electrically switching the electrodes of the at least one droplet generation channel to separate droplets of the cell suspension from the cell suspension in the at least one reservoir region;
(3) Performing power-on and power-off control on electrodes of at least one droplet generation channel and the single cell capture area to enable the cell suspension droplets to be displaced to the hydrophilic part of the single cell capture area, enabling a single cell in the cell suspension droplets to face the hydrophilic part, then performing power-off standing to enable the single cell to naturally settle to the bottom of the hydrophilic part under the action of gravity, and then removing the cell suspension droplets to enable the single cell to remain at the bottom of the hydrophilic part;
(4) De-energizing the electrodes of at least one of the reservoir zones and washing the remaining cell suspension therein with sterile water;
(5) Carrying out on-off control on electrodes of at least one liquid storage area, at least one liquid drop generation channel and a single cell capture area, conveying cell lysate to a hydrophilic part with single cells settled at the bottom from the at least one liquid storage area, carrying out cell lysis reaction with the single cells, heating by a heating device to fully release miRNA from the cell lysate, carrying out on-off control on the electrodes of the single cell capture area and a plurality of library reaction areas, and conveying materials positioned in the hydrophilic part to the corresponding library reaction areas;
(6) According to the step (5), repeatedly carrying out on-off control on electrodes of at least one liquid storage area, at least one liquid drop generation channel, a single cell capture area and a plurality of library reaction areas, sequentially moving a 3 'joint connection reagent, an excess 3' joint digestion reagent, a5 'joint connection reagent, a first reverse transcription pre-reaction reagent, a second reverse transcription pre-reaction reagent and a reverse transcription reagent from the at least one liquid storage area to the corresponding library reaction areas, and sequentially completing a 3' joint connection reaction, an excess 3 'joint digestion reaction, a 5' joint connection reaction, a first reverse transcription pre-reaction, a second reverse transcription pre-reaction and a reverse transcription reaction by heating through a heating device;
(7) Adding an amplification reagent into a material obtained after completion of reverse transcription reagents in a plurality of library reaction regions, and after fully mixing, carrying out a first amplification reaction by a gene amplification device;
(8) And (4) mixing the materials from different library reaction regions obtained in the step (7) with miRNA library construction amplification reaction reagents respectively, and carrying out a second amplification reaction through an amplification device to obtain miRNA sequencing libraries corresponding to different single cells.
2. The method of claim 1, wherein: the formula of the cell lysate is as follows:
3. the method of claim 1, wherein: the formula of the 3' joint connecting reagent is as follows:
4. the method of claim 1, wherein: the formula of the redundant 3' joint digestion reagent is as follows:
5. the method of claim 1, wherein: the formula of the 5' joint connecting reagent is as follows:
6. the method of claim 1, wherein: the formula of the first reverse transcription pre-reaction reagent is as follows:
the formula of the second reverse transcription pre-reaction reagent is as follows:
the formula of the reverse transcription reaction reagent is as follows:
7. the method of claim 1, wherein: the formula of the amplification reagent is as follows:
8. the method of claim 1, wherein: the miRNA library construction amplification reaction reagent comprises the following formula:
9. the method of claim 1, wherein: the hydrophilic part is a circular groove which is formed by adopting a local stripping technology, has a hydrophilic surface and is 90-110 mu m in diameter.
10. The production method according to any one of claims 1 to 9, characterized in that: the number of the liquid storage areas is two, the number of the liquid drop generating channels is two, and the single-cell capturing area is provided with seven hydrophilic parts.
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