CN114854577A

CN114854577A - Single cell transcriptome sequencing library construction platform and use method thereof

Info

Publication number: CN114854577A
Application number: CN202110154908.8A
Authority: CN
Inventors: 杨朝勇; 许醒; 张倩倩; 杨健; 蔡林峰
Original assignee: Xiamen Deyun Xinzhun Technology Co ltd
Current assignee: Xiamen Deyun Xinzhun Technology Co ltd
Priority date: 2021-02-04
Filing date: 2021-02-04
Publication date: 2022-08-05

Abstract

The invention relates to a single-cell transcriptome sequencing library construction platform, which comprises a digital microfluidic chip, a circuit control module, a controller, a magnetic separation control module of a magnet and a cam positioned at the bottom of the magnet and used for adjusting the magnet to ascend and descend, wherein the circuit control module is connected with the digital microfluidic chip; the chip comprises an upper polar plate and a lower polar plate, wherein the lower polar plate comprises a substrate, an electrode layer, a dielectric layer and a hydrophobic layer, and the upper polar plate comprises the hydrophobic layer, a grounding conductive layer and a top plate; the lower plate is divided into a liquid drop driving area and a plurality of liquid storage areas; the hydrophobic layer comprises hydrophilic dots; the electrode layer comprises a driving electrode array and a plurality of liquid storage electrode arrays. The invention also relates to methods of using such library construction platforms. The construction platform and the use method of the invention realize full-automatic reagent control and addition, flexible, rapid capture of single cells, sensitive, accurate and stable gene detection, and low cost. The integrated preparation platform greatly simplifies the experimental mode and steps, and makes the detection and analysis of the high-throughput cell genetic information possible.

Description

Single cell transcriptome sequencing library construction platform and use method thereof

Technical Field

The invention relates to the field of digital microfluidic chips, in particular to a single-cell transcriptome sequencing library construction platform and a use method thereof.

Background

Cells are the basic unit of life. From the perspective of a single organism, genes within each cell are essentially identical, but gene expression is highly heterogeneous, thereby constituting diversity in tissues, organs, and systems. This intercellular heterogeneity occurs even in cells of the same type. Differences in gene expression between cells have a critical impact on biological processes such as embryonic development, cancer growth, immune response, and the like. The population-level-based transcriptome analysis technology cannot reveal information of heterogeneity among cells, so that the development of single-cell transcriptome analysis technology is necessary.

Based on the second generation sequencing technology, the gradually developed single cell RNA sequencing (scRNA-seq) method researches the expression type and level of mRNA at the single cell level, and becomes an important tool for revealing rare cells, distinguishing cell subsets and constructing cell spectrograms. The conventional process comprises single cell capture, single cell lysis, mRNA reverse transcription, cDNA amplification, purification, library construction and sequencing on a computer. At present, scRNA-seq is mainly divided into two methods, including a tag-based sequencing method and a full-length-based sequencing method. Among these, tag-based scRNA-seq (e.g., Drop-seq, Indorps, sci-RNA-seq, Paired-seq, etc.) allows for high throughput analysis by tagging the cDNA of each cell using cell barcode technology, but there is an analytical bias at either the 3 'or 5' end, and therefore fewer variable truncations and SNPs can be detected. And it is difficult to identify the primitive cells by this method due to the mixing operation. The single cell is operated one by one based on the full-length scRNA-seq method (such as Smart-seq2), the flux is small, but the method has high detection capability on the variable shearing of genes and SNPs, and is easy to locate the source cell, so the method still has the advantages in the aspect of gene expression detection of the given cell.

However, current full-length based scRNA-seq methods are mainly performed in centrifuge tubes, require large reaction volumes, cumbersome manual operations, which result in increased reagent consumption, increased contamination potential, and to some extent reduced sensitivity and accuracy of the method. The microfluidic technology has the characteristics of integrated operation platform, small reaction volume and closed reaction space, so that the microfluidic device has the advantages of low reagent consumption, low pollution probability, and high sensitivity and accuracy. The Beijing university research group develops an integrated micro-fluidic chip for full-length scRNA-seq analysis, and the method improves the accuracy and sensitivity of gene expression measurement by using different reaction chambers to carry out different reactions due to the characteristics of small volume and closed type. A similar design is used by the commercial Fluidigm C1 system, which is now compatible with CEL-seq, Smart-seq, etc. to provide integrated operation. The university of virginia university of studios research group designed a microfluidic device for Smart-seq2 experiments that performed reagent removal and delivery by concentration gradient driven diffusion, which procedure could integrate all processes into one chamber. However, in the above microfluidic platforms, a micro pump/valve structure is generally added to the bottom layer of the chip to control the flow of the reagent, and complicated chip structure design, manufacturing technology and strict flow control are required. Moreover, these platforms do not allow for selective cell capture, and are difficult to remove unhealthy or dead cells, resulting in some wasted space and reagents. In addition, all current microfluidic platforms can only complete single cell capture, single cell lysis, mRNA reverse transcription and cDNA amplification, and subsequent purification and library construction processes still need to be transferred to a centrifuge tube for carrying out due to the need of complex magnetic separation and other operations. Therefore, a simple, sensitive and accurate single-cell transcriptome sequencing platform is still lacked at present, and the construction of an integrated single-cell transcriptome sequencing library of 'cell in and library out' is realized.

Digital Microfluidics (DMF) is a microfluidic technology that uses an array of electrodes to automatically manipulate discrete droplets between two spaced parallel plates. DMF is based on the principle of dielectric wetting (EWOD), and applies a sequence of voltages to an external electrical control system, so that droplets can be distributed, combined, split and transported along a pre-designed path. Compared with the traditional microfluidics, the DMF has the advantages of less sample demand and strong parallel operation capability, and does not need to depend on a pump/valve and a three-dimensional fluid channel, so that reagents can be flexibly added. This automation, flexibility and convenience of fluidic manipulation has led to DMF promising applications in complex and repetitive biological and chemical analyses, such as immunoassays, nucleic acid detection, cell measurements. However, the conventional DMF chip still cannot integrate cell capture, detection, purification and library construction, and it is difficult to provide a fully automatic integrated experimental platform.

In this regard, we hope to combine DMF technology and magnetic separation technology to provide a full-automatic single-cell transcriptome sequencing library construction platform, which integrates operations of single-cell capture, single-cell lysis, mRNA reverse transcription, cDNA amplification, purification, library construction, etc., and can realize rapid, sensitive and accurate construction of a "cell-in, library-out" single-cell transcriptome sequencing library.

Disclosure of Invention

Aiming at the problems in the prior art, the project aims to realize the rapid, sensitive and accurate construction of the single cell transcriptome sequencing library from cell entrance and library exit by integrating the operations of single cell capture, single cell lysis, mRNA reverse transcription, cDNA amplification, purification, library construction and the like through a full-automatic single cell transcriptome sequencing library construction platform.

In order to achieve the purpose, the invention discloses a single-cell transcriptome sequencing library construction platform, which comprises a digital microfluidic chip, a circuit control module, a controller and a magnetic separation control module;

the magnetic separation control module is positioned at the lower part of the digital microfluidic chip and is used for generating a magnetic field so as to facilitate DNA purification by using magnetic beads on a platform. The magnetic separation control module comprises a magnet and a cam which is positioned at the bottom of the magnet and used for adjusting the magnet to ascend and descend;

the digital microfluidic chip comprises an upper polar plate and a lower polar plate which are oppositely arranged in parallel, and the upper polar plate and the lower polar plate are separated by a space for liquid to move; the lower electrode plate sequentially comprises a substrate, an electrode layer, a dielectric layer and a hydrophobic layer from bottom to top, and the upper electrode plate sequentially comprises the hydrophobic layer, a grounding conductive layer and a top plate from bottom to top;

the lower plate is divided into a liquid drop driving area and a plurality of liquid storage areas according to the function of the electrode;

the hydrophobic layer comprises a plurality of hydrophilic points positioned in the liquid drop driving area to form micropores, can be used for single cell capture, lysis, mRNA reverse transcription, cDNA amplification, purification, library establishment and other operations, and achieves the same or similar effects as the micropores of the traditional chip or a capture chamber;

the electrode layer comprises a driving electrode array positioned in the liquid drop driving area and a liquid storage electrode array respectively positioned in the liquid storage areas;

the circuit control module comprises a plurality of electrode access ports and leads for respectively connecting the electrode access ports with the electrode arrays in the digital microfluidic chip;

the plurality of electrode access ports are further connected to a digital microfluidic control instrument, and the control instrument drives liquid drops in the chip to move by applying voltage between a top plate and a bottom plate of the digital microfluidic chip. In some embodiments, the circuit control module further comprises an electrode thimble connector for connecting the driving electrode array with a lead and further accessing the digital microfluidic controller. The manner in which the array of drive electrodes is connected to the control unit is well known in the art and can be adjusted by one skilled in the art according to the actual needs.

The magnet is introduced in the present invention to separate the magnetic beads from the supernatant, allowing the purification and pooling step to be integrated on the DMF chip. In the scheme of the invention, the top of the magnet can correspond to one or more electrodes of the microfluidic chip to be introduced with the magnetic beads. When the magnetic bead separation device is used, the magnet is lifted to the bottom of the position, corresponding to the magnetic bead, on the chip through the control cam, and then the driving electrode is electrified, so that the supernatant is moved to an adjacent empty electrode from the electrode containing the magnetic bead, and the magnetic bead separation is realized.

In some embodiments, the magnet is a permanent magnet or an electromagnet. When the magnet is a permanent magnet, the shape thereof may be a bead shape, a needle shape, a columnar shape, or the like. In some embodiments, the magnet is cylindrical.

In some embodiments, the ratio of the magnet diameter to the maximum electrode diameter may be 0.5 to 10. In some embodiments, the magnet is at a vertical distance of less than 4cm from the bottom of the chip.

In some embodiments, the magnetic separation control module further comprises a cam located at the bottom of the magnet and used for adjusting the magnet to ascend and descend, the position of the magnet is adjusted through the cam, the generation and removal of a magnetic field can be controlled, and the control of magnetic beads in the platform is realized, so that the steps of magnetic bead incubation, magnetic bead adsorption, magnetic bead cleaning, magnetic bead elution and the like in the purification process are completed.

In some embodiments, the droplet driving region of the lower plate further comprises a reaction region and a plurality of droplet generation channels for connecting the reaction region with the reservoir region for splitting droplets from the liquid in the reservoir region for subsequent reactions.

In some embodiments, the drive electrode array comprises an array of reaction electrodes and an array of droplet generation channel electrodes positioned to correspond to the reaction zone and droplet generation channel; the droplet generation channel electrode array is used for connecting the reaction electrode array with the reservoir electrode array.

In some embodiments, the number of hydrophilic sites is 1, 2, 3, or more.

In some embodiments, the shape of the hydrophilic spot is circular, square, crescent, irregular.

In some embodiments, the hydrophilic sites are located in the reaction zone of the lower plate.

In some embodiments, the hydrophilic spots are formed by applying techniques such as local lift-off in a droplet driving region, particularly a reaction region, of the hydrophobic layer. Specific partial peeling techniques are well known to those skilled in the art.

In the invention, after the cell suspension liquid drop is driven by the manipulator to reach the hydrophilic point micropore, the cell suspension liquid drop quickly forms a sub-liquid drop containing a single cell in the micropore under the action of surface tension. More specifically, when a cell suspension drop reaches a hydrophilic point pore, a small drop is formed when a large drop passes through a hydrophilic region under the action of surface tension, namely, passive dispensing. In addition, cells are hydrophilic and therefore tend to remain within the hydrophilic region. When the concentration of cells in the droplet is 10 ⁵ Below one/mL, a single cell may be left in the hydrophilic region. If no single-cell droplets are generated, the large droplets can be moved back to the hydrophilic region, and the above steps are repeated until a single-cell sub-droplet is obtained.

In some embodiments, the reservoir region further comprises one or more of a cell suspension reservoir region, a lysate reservoir region, a reverse transcription reagent reservoir region, a PCR amplification reagent reservoir region, a magnetic bead reagent reservoir region, and/or a waste region. Each reservoir region is used for loading corresponding liquid for subsequent treatment of cell capture, and the waste region is used for collecting liquid which is not needed in the reaction process. Wherein, the lysate storage area and the magnetic bead reagent storage area can be shared.

In some embodiments, the reservoir electrode array includes a cell suspension reservoir electrode array, a lysate reservoir electrode array, a reverse transcription reagent reservoir electrode array, a PCR amplification reagent reservoir electrode array, a magnetic bead reagent reservoir electrode array, and a waste liquid electrode array at a location corresponding to the reservoir region.

In some embodiments, each of said electrode arrays consists of at least 1 electrode unit. In some preferred embodiments, the reaction electrode array is composed of 8 to 32 electrode units, preferably 24 electrode units. And the reaction electrode units are preferably connected in series and assembled in a plurality of rows (e.g., 1 row, 2 rows, 3 rows, or 4 rows) to form a complete quadrilateral planar structure. In some preferred embodiments, each of the reservoir electrode arrays consists of 1-3 electrode units, preferably 1 electrode unit. In some preferred embodiments, the number of droplet generation channel electrode arrays is the same as the number of reservoir electrode arrays. In some more preferred embodiments, each of said droplet generation channel electrode arrays consists of 1-5, in particular 2 or 3, electrode units, respectively.

In some embodiments, the size of each electrode unit in different electrode arrays is the same. In some embodiments, the size of each electrode unit in different electrode arrays is partially the same. In some embodiments, the size of each electrode unit in different electrode arrays is different. Preferably, the electrode unit size is selected according to the specific reaction reagent volume.

In some embodiments, each electrode unit is a quadrilateral, and the sides of the quadrilateral are linear or zigzag, preferably zigzag, so that when a liquid drop stays at a certain electrode, the liquid drop can contact the surrounding electrode to facilitate driving. In other embodiments, the electrode units are circular or triangular in shape.

In some embodiments, the edges of two adjacent electrode units are fitted to each other. In a general aspect, the electrode gap is greater than 10 μm. Preferably, the electrode gap is in the range of 13-17 μm, for example 15 μm, to obtain an optimally sized electrode.

In some embodiments, the electrode unit is square-like with jagged sides. In some embodiments, the electrode unit is rectangular-like with jagged sides.

In some embodiments, each electrode unit in the array of drive electrodes may have a side length of 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, etc. for dispensing droplets of various volumes. In some embodiments, each electrode unit in the reservoir electrode array can have a side length of 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2.0mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3.0mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4.0mm, and the like. More preferably, the size of each electrode unit in the reservoir electrode array may be 1.8mm by 1.2mm, 2.4mm by 2.4mm or 3.6mm by 2.4 mm.

In some embodiments, an oil phase is filled between the upper plate and the lower plate to prevent the liquid drops from volatilizing. Preferably, the oil phase is a silicone oil, such as PMS-200.

In some embodiments, the bottom plate of the microfluidic chip of the present invention may be made of glass, printed circuit board, or paper base; the dielectric layer can be made of photoresist, PDMS, paralyene-C, Si ₃ N ₄ (ii) a The hydrophobic layer can be Teflon or polytetrafluoroethylene PTFE aqueous dispersion; the hydrophobic layer material of the lower plate and the hydrophobic layer material of the upper plate can be the same or different. The material of the top plate ground conductive layer may be Indium Tin Oxide (ITO), which is often coated directly on the glass as the top plate.

The specific method of applying a time-sequential voltage to an electrode unit on a DMF chip by an external electrical signal control system based on the dielectric wetting principle to drive a droplet to precisely move along a preset path is known in the art. According to the technology, in the digital microfluidic chip, the electrode driving circuit is controlled, the on-off control is carried out on the electrode where the liquid drop is located or the peripheral electrode according to the preset sequence, the electric field is applied to enable the liquid surface to generate polarity hydrophilicity, the surface tension is changed, the liquid surface is flattened, the polarization position is controlled to generate a tension gradient, and then the controlled liquid drop is enabled to be displaced on the surface of the microfluidic platform. Finally, the generation, movement, combination and division of the liquid drops are realized. The specific principle can be referred to textbook "digital microfluidic technology and application" (scientific press; Yang Dynamo, Yuan Qingyu).

In another aspect, the present invention provides a method for using a single-cell transcriptome sequencing library construction platform, comprising:

a. the cell suspension, lysate and reverse transcription reagent are added to the reservoir region first, and pre-energization is performed in the corresponding reservoir region.

b. The electrode driving circuit is controlled by the controller and the circuit control module, the on-off control of the electrodes is carried out according to a preset sequence, so that a cell suspension liquid drop is generated from the electrode unit of the liquid storage area, and the liquid drop is moved to the hydrophilic point area by controlling the driving electrode array connected with the cell suspension liquid drop. This process is repeated to load each hydrophilic site region with a droplet of cell suspension.

c. By applying a voltage to adjacent electrodes, the position of the cell in the droplet is adjusted so that a single cell is located above the hydrophilic site. And powering off the driving electrode at the hydrophilic point, and waiting to allow the cells to settle under the action of gravity.

d. And controlling an electrode driving circuit, applying voltage to adjacent electrodes of the hydrophilic sites to enable the cell suspension liquid drops to pass through the hydrophilic sites, and leaving small drops on the hydrophilic sites under the action of surface tension in the process so as to generate small drops containing single cells. This step can be repeated by continuing mixing and splitting of the primary and small droplets until the target cell is obtained. The process was repeated so that each hydrophilic site area was loaded with droplets containing a single cell.

e. And controlling the electrode driving circuit to move the original cell suspension liquid drops to the waste liquid area for removal.

f. And controlling an electrode driving circuit to carry out on-off electric control electrode unit manufacturing of the electrodes according to a preset sequence, so that a lysate liquid drop is generated from the liquid storage area and moves to a hydrophilic point area to be mixed with the small liquid drop containing the single cell. This process is repeated so that each hydrophilic site area is loaded with a lysate droplet and mixed with a droplet containing a single cell.

g. The chip was placed on a heating plate to lyse the cells sufficiently.

h. And controlling the electrode driving circuit to perform on-off control of the electrodes according to a preset sequence, so that a reverse transcription reagent droplet is generated from the liquid storage area and moves to the hydrophilic locus area to be mixed with the droplet containing the cell lysate. This process is repeated so that each hydrophilic site region is loaded with a droplet of reverse transcription reagent and mixed with a droplet containing cell lysate.

i. Completing reverse transcription reaction of mRNA of single cells to generate cDNA.

j. Adding PCR amplification reagent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on the electrodes according to a preset sequence, so that a PCR amplification reagent liquid drop is generated from the liquid storage area and moves to a hydrophilic locus area to be mixed with the liquid drop containing cDNA. This process is repeated so that each hydrophilic site region is loaded with a droplet of cDNA amplification reagent and mixed with a droplet containing cDNA product.

k. And carrying out PCR amplification on the single cells in the chip.

And l, adding the purified magnetic bead reagent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that a purified magnetic bead liquid drop is generated from an electrode unit of the liquid storage area, moves to a hydrophilic locus area, and is mixed with a liquid drop containing a cDNA amplification product. This process is repeated so that each hydrophilic site region is loaded with a droplet of purified magnetic beads and mixed with a droplet containing cDNA amplification products.

And m, after standing, introducing a magnetic field, controlling an electrode driving circuit, performing on-off control on electrodes according to a preset sequence, moving a supernatant part in the liquid drop to a waste liquid area, removing, keeping a magnetic bead part, and removing the magnetic field.

And n, adding the eluent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on the electrodes according to a preset sequence, so that eluent liquid drops are generated from an electrode unit of the liquid storage area, move to a magnetic bead area and are mixed with the magnetic beads. This process is repeated so that each bead region is loaded with a droplet of eluent and mixed with the beads.

And o, after standing, introducing a magnetic field, controlling an electrode driving circuit, performing on-off control on the electrodes according to a preset sequence, and moving the supernatant part in the liquid drop to an adjacent electrode.

And p, removing the magnetic field, controlling an electrode driving circuit, performing power-on and power-off control on the electrodes according to a preset sequence, and transferring the magnetic beads to a waste liquid area.

And q, starting to perform a library building reaction. The library building reaction comprises a fragmentation reaction, a termination reaction and an amplification reaction. Fragmentation reagents were first added to the stock region. And controlling an electrode driving circuit to perform on-off control on the electrodes according to a preset sequence, so that a fragmented reaction droplet is generated from the liquid storage region electrode unit, moves to the supernatant position in the step 15 and is mixed with the supernatant droplet. This process is repeated to mix each fragmented reagent droplet with each supernatant droplet.

And r, fragmenting the single-cell cDNA in the chip to generate a cDNA fragmentation product.

s, adding a reaction termination reagent to the liquid storage region, controlling an electrode driving circuit, and performing on-off control of electrodes according to a preset sequence, so that a reaction termination liquid drop is generated from an electrode unit of the liquid storage region, moved to a liquid drop position containing a cDNA fragmentation product and mixed with the liquid drop position. This process is repeated so that each of the droplets of the termination reaction reagent is mixed with each of the cDNA-containing fragmentation products and left to stand.

And t, adding an amplification reaction reagent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on the electrodes according to a preset sequence, so that a fragmentation reaction liquid drop is generated from an electrode unit of the liquid storage area, moves to the position of the liquid drop in the previous step and is mixed with the fragmentation reaction liquid drop. This process is repeated to mix each droplet of amplification reagent with each droplet of the previous step.

u. performing PCR amplification on the single cell cDNA fragmentation product in the chip, and adding a sequencing adaptor.

v. repeating the steps of 12-15 to purify the product, and finally obtaining the single-cell transcriptome library product. The product was taken off the chip and directly subjected to the second generation sequencing of illumina.

The invention has the beneficial effects that:

1. the construction platform of the invention realizes full-automatic reagent control and addition. In the existing method, the operation of the centrifuge tube needs to manually add the reagents in sequence, which is time-consuming, labor-consuming and tedious; the micro-fluidic chip is still complex in reagent addition because an external pump device and micro-valve micro-pump control inside the chip need to be combined. Based on the full-automatic droplet control capability of DMF, sequential addition of cell suspension, lysate, reverse transcription reagent, PCR reagent and the like on a DMF chip can be realized through programmed design, so that the complexity, time and labor consumption of centrifuge tube operation in the existing method are overcome, and the complex pump valve operation and flow rate control of the current microfluidic platform are overcome.

2. The construction platform of the invention realizes flexible and rapid capture of single cells. The existing method mainly adopts the technologies of limiting dilution, microdissection and capillary selection during single cell separation, has the disadvantages of complex operation, time and labor consumption and high damage to cells. Although there is a single cell capture technology based on microfluidic chip, it requires complicated structure design and micro valve micropump control (such as designing cell card slot, etc.), the operation is still troublesome, and once the unwanted cells are captured, it is difficult to remove the unwanted cells. The digital microfluidic chip of the construction platform of the invention introduces hydrophilic sites as micropores in the hydrophobic layer, and when cell suspension flows through the hydrophilic sites, droplets containing single cells can be left in the hydrophilic sites under the action of surface tension. And by electrical control, it is also possible to remove the originally captured cells by mixing the primary cell suspension with the hydrophilic site droplets again, and then repeating the same process to generate new droplets containing single cells. Therefore, the rapid flexible high-throughput capture of single cells can be realized. The capture time of single cells was within 30 s.

3. The construction platform of the invention realizes sensitive gene detection and has low cost. In the existing method, the reaction volume of the centrifuge tube operation is microliter level, and the reagent consumption cost is high. In addition, the reaction efficiency is limited at such volumes. As the DMF chip can realize the droplet control of nano-upgrading, the concentration of a reaction template (the mRNA of a single cell) is greatly improved in a reaction space of nano-upgrading, thereby increasing the reaction rate. Meanwhile, the surface of the DMF chip is basically hydrophobic, so that the adsorption of a template and a subsequent reaction product is reduced, and the recovery rate of the product is increased. Both aspects make the gene detection more sensitive. Compared with the operation of a centrifuge tube, the consumption of the reagent can be reduced by about one hundred times. The construction of a single-cell transcriptome sequencing library in the centrifuge tube operation needs at least 300 elements; the reagent consumption cost of the single-cell transcriptome sequencing library on the DMF chip is only about 3 yuan/piece, and the cost is greatly reduced.

4. The construction platform of the invention realizes accurate gene detection. In the existing method, the centrifuge tube always needs to be opened continuously for reagent addition during operation, errors caused by human factors are large, and external pollution is easily introduced, so that the accuracy of gene detection is easily reduced. The DMF chip has good uniformity when generating the liquid drops, and can overcome the reagent addition difference caused by manual operation of a centrifuge tube, thereby ensuring the stability of the volume of the reaction liquid drops. Meanwhile, the surface of all the liquid drops is covered with a layer of silicone oil, so that external pollution can be isolated. Both aspects increase the stability of the platform in gene detection.

5. The invention realizes an integrated unicellular transcriptome sample preparation platform of 'cell entering and sample exiting'. In the existing method, no micro-fluidic chip platform can realize the preparation of the integrated single-cell transcriptome library of cell inlet and sample outlet. A magnetic control module is introduced into the construction platform, and magnetic bead control is realized by combining a liquid drop control technology under the action of a magnet, so that the steps of magnetic bead incubation, magnetic bead adsorption, magnetic bead cleaning, magnetic bead elution and the like in the purification process can be completed. Meanwhile, the library building step is also integrated, and the processes of product fragmentation, index addition, amplification and the like are completed by introducing various reagents in the library building step. The finally generated library can be directly used for computer. Therefore, the platform can realize the preparation of the integrated single-cell transcriptome library of the cell inlet and the cell outlet, greatly simplifies the experimental mode and the steps and makes the detection and analysis of the high-throughput cell genetic information possible.

Drawings

FIG. 1 is a schematic diagram of a single cell transcriptome sequencing library construction platform in an embodiment of the present invention.

Fig. 2 is a cross-sectional structure diagram (cross section) and a partial structure enlarged view of the digital microfluidic chip according to the embodiment of the present invention.

Fig. 3 is a CAD design top view of a digital microfluidic chip and a circuit control module according to an embodiment of the invention.

Fig. 4 is a diagram of a digital microfluidic chip and a circuit control module according to an embodiment of the present invention.

FIG. 5 is a flow chart of single cell separation using a platform constructed according to an embodiment of the present invention (FIG. 5a. cell suspension introduction; FIG. 5b. single cell sedimentation at hydrophilic sites; FIG. 5c. single cell separation).

FIG. 6 is a characterization of droplet uniformity for droplet generation using a build platform according to an embodiment of the present invention.

FIG. 7 is a drop mixing efficiency characterization of drop mixing using an example build platform of the invention.

Detailed Description

The technical scheme of the invention is described in detail in the following with reference to the accompanying drawings. Although the present invention has been described in detail with reference to the preferred embodiments, it will be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the present invention, which is defined in the claims.

Example 1

The following describes in detail the single cell transcriptome sequencing library construction platform according to the present invention with reference to fig. 1 to 5, which comprises a digital microfluidic chip 100, a circuit control module 200, a controller 300, and a magnetic separation control module 400.

The magnetic separation control module 400 is located at the lower part of the digital microfluidic chip 100 and is used for generating a magnetic field to facilitate DNA purification using magnetic beads on a platform. The magnetic separation control module 400 includes a magnet 410 and a cam 411 controlling the up and down movement of the magnet.

The digital microfluidic chip 100 further includes a lower plate 120 and an upper plate 110 disposed opposite to each other in parallel, and spaced apart therefrom for movement of liquid; the lower plate 120 sequentially includes a bottom plate 121, an electrode layer 122, a dielectric layer 123 and a hydrophobic layer 124 from bottom to top, and the upper plate 110 sequentially includes a hydrophobic layer 111, a grounded conductive layer 112 and a top plate 113 from bottom to top. In some embodiments, the grounded conductive layer 112 is coated directly on the top plate 113.

The lower plate 120 is divided into a droplet driving region 1 and a plurality of liquid storage regions 2 according to the function of electrodes.

The hydrophobic layer 124 comprises a plurality of hydrophilic spots 124A in the droplet driving region 1 to form micropores, which can be used for single cell capture, and can achieve the same or similar effect as the micropores of a conventional chip, or referred to as capture chambers. In a preferred embodiment, 2 hydrophilic spots are provided.

The electrode layer 122 includes a driving electrode array 3 located in the droplet driving region 1, and a reservoir electrode array 4 located in the plurality of reservoir regions 2, respectively.

The circuit control module 200 includes a plurality of electrode inlets 210 and leads 211 respectively connecting the electrode inlets with the electrode array in the digital microfluidic chip.

The plurality of electrode accesses 210 are further connected to a digital microfluidic controller 300 that drives the movement of the droplets within the chip by applying a voltage between the top plate 113 and the bottom plate 121 of the digital microfluidic chip 100.

The circuit control module 200 further includes an electrode thimble connector 212 for connecting the driving electrode array 3 with a lead 211, and further accessing the digital microfluidic controller 300 through the electrode access 210.

The magnet 410 is a permanent magnet or an electromagnet. When the magnet is a permanent magnet, the shape thereof may be a bead shape, a needle shape, a columnar shape, or the like. In a preferred embodiment, the magnets are cylindrical.

The diameter ratio of the magnet 410 to the active electrode of the chip is 0.5 to 10.

Magnetic separation control module 400 still can be including being located magnet 410 bottom and being used for adjusting magnet 410 and ascending cam 411 with descending, through the position of cam 411 regulation magnet 410, the production and the removal of steerable magnetic field realize controlling of magnetic bead in the platform to accomplish steps such as magnetic bead incubation, magnetic bead absorption, magnetic bead washing and magnetic bead elution in the purification process.

The drop drive zone 1 of the lower plate further comprises a reaction zone 5 and a plurality of drop generating channels 7. The droplet generation channel 7 is used for connecting the reaction region 5 with the liquid storage region 2, and is used for splitting droplets for subsequent reaction from the liquid in the liquid storage region 2.

The drive electrode array 3 comprises a reaction electrode array 8 and a droplet generation channel electrode array 9 positioned to correspond to the reaction zone 5 and the droplet generation channel 7; a droplet generation channel electrode array 9 is used to connect the reaction electrode array 8 with the reservoir electrode array 4.

The number of the hydrophilic dots 124A is 1, 2, 3 or more, and in this embodiment, the build platform is provided with 2 hydrophilic dots. And the hydrophilic spot 124A is located in the reaction region 5 of the lower plate 120.

Preferably, the shape of the hydrophilic dots 124A is circular, square, crescent, irregular

The liquid storage area 2 further comprises a cell suspension liquid storage area 10, a lysate liquid storage area 11, a reverse transcription reagent liquid storage area 12, a PCR amplification reagent liquid storage area 13, a magnetic bead reagent liquid storage area 14 and a waste liquid area 15. Each reservoir region is used for loading corresponding liquid for subsequent treatment of cell capture, and the waste region is used for collecting liquid which is not needed in the reaction process.

The liquid storage electrode array 4 comprises a cell suspension liquid storage electrode array 16, a lysate liquid storage electrode array 17, a reverse transcription reagent liquid storage electrode array 18, a PCR amplification reagent liquid storage electrode array 19, a magnetic bead reagent liquid storage electrode array 20 and a waste liquid electrode array 21 at the corresponding positions of the liquid storage area 2.

In some embodiments, the lysate reservoir region and the magnetic bead reagent reservoir region share the same region, and the lysate reservoir electrode array and the magnetic bead reagent reservoir electrode array share the same electrode array.

Each of the electrode arrays is composed of at least 1 electrode unit. Preferably, the reaction electrode array is composed of 8-32 electrode units, more preferably 24 electrode units. And the reaction electrode units are preferably connected in series and assembled in a plurality of rows (e.g., 1 row, 2 rows, 3 rows, or 4 rows) to form a complete quadrilateral planar structure. Each liquid storage electrode array is respectively composed of 1-3 electrode units, preferably 1 electrode unit. The number of electrode arrays of the droplet generation channels is the same as that of the electrode arrays of the liquid storage channels. More preferably, each droplet generation channel electrode array is composed of 1-5, in particular 2 or 3 electrode units, respectively.

The size of each electrode unit in different electrode arrays is the same, partially the same or different.

In various embodiments, each electrode unit may have a quadrilateral shape with straight or serrated edges, preferably serrated.

The edges of two adjacent electrode units are tightly connected through mutual fit.

Preferably, the electrode unit is square-like or rectangular-like with zigzag sides.

The side length of each electrode unit in the drive electrode array 3 may be 0.6mm, 0.7mm, 0.8mm, 0.9mm, 1.0mm, 1.1mm, 1.2mm, etc. for dispensing droplets of various volumes. The side length of each electrode unit in the reservoir electrode array 4 may be 1.0mm, 1.1mm, 1.2mm, 1.3mm, 1.4mm, 1.5mm, 1.6mm, 1.7mm, 1.8mm, 1.9mm, 2.0mm, 2.1mm, 2.2mm, 2.3mm, 2.4mm, 2.5mm, 2.6mm, 2.7mm, 2.8mm, 2.9mm, 3.0mm, 3.1mm, 3.2mm, 3.3mm, 3.4mm, 3.5mm, 3.6mm, 3.7mm, 3.8mm, 3.9mm, 4.0mm, etc. More preferably, the size of each electrode unit in the reservoir electrode array 4 may be 1.8mm by 1.2mm, 2.4mm by 2.4mm or 3.6mm by 2.4 mm.

An oil phase is filled between the upper plate 110 and the lower plate 120 to prevent the liquid droplets from volatilizing. Preferably, the oil phase is a silicone oil, such as PMS-200.

The bottom plate 121 can be made of glass, printed circuit board, or paper base; the material of the dielectric layer 123 may be photoresist, PDMS, paralyene-C, Si ₃ N ₄ (ii) a The hydrophobic layer 124 may be teflon or polytetrafluoroethylene PTFE aqueous dispersion; the lower plate hydrophobic layer 124 and the upper plate hydrophobic layer 125 may be the same or different materials. The material of the upper plate grounding conductive layer 112 may be Indium Tin Oxide (ITO)It is often coated directly on the glass as the top plate 113.

The platform in this embodiment can be manufactured and built in the following manner:

1. designing an electrode layer pattern: designing a chip by using a software CAD, wherein a design drawing is shown in FIG. 6, a chip real object is shown in FIG. 7, and a reservoir electrode array comprises 5 sub-reservoir electrode arrays with areas of 1.8mm multiplied by 1.2mm, 2.4mm multiplied by 2.4mm and 3.6mm multiplied by 2.4mm respectively, and is used for storing reagents; the drive electrode array comprises 36 drive electrodes for droplet drive (including generation, movement, splitting, merging), which specifically comprises 6 small electrodes (0.6mm x 0.6mm), 26 medium electrodes (0.8mm x 0.8mm) and 4 large electrodes (1.2mm x 1.2 mm).

2. Manufacturing a lower polar plate:

(a) evaporating a 300-nanometer thick chromium layer on a lower plate glass substrate by using a magnetron sputtering method, and forming a chromium electrode array with a specific structure by wet etching;

(b) forming a dielectric layer by using photoresist in a spin coating mode;

(c) and forming a local circular hydrophilic site in a reaction area of the hydrophobic layer by using a polytetrafluoroethylene PTFE aqueous dispersion through a local stripping and alignment technology.

3. Manufacturing an upper polar plate:

(a) depositing a grounded conductive layer material on the top plate by deposition;

(b) the hydrophobic layer is formed by a spin-on annealing process using an aqueous dispersion of polytetrafluoroethylene PTFE.

4. Two layers of adhesive tapes are adhered to two sides of the lower polar plate of the chip, the height is 2 multiplied by 60 mu m, then the upper polar plate is pressed on the lower polar plate, a gap with certain thickness is formed between the upper polar plate and the lower polar plate, a sandwich structure is formed between the upper polar plate and the lower polar plate and the liquid drops in the gap, and silicon oil is filled between the two plates. And (4) forming a hole at the upper polar plate position above the liquid storage area of the lower polar plate, so that liquid is introduced through automatic sample injection.

5. The electrode on the lower electrode plate is connected with a DMF manipulator through a lead and an electrode access port, and the sine wave potential is set to be (150-.

6. By using the most strongly magnetic rubidium-iron-boron magnet in the permanent magnet as the magnetic main body, the magnetic field is timely turned on and removed through a blending system of control software. The specific operation is as follows: when the liquid drops are controlled to move right above the columnar magnet, the motor drives the cam to rotate, the magnet is pushed upwards, and the magnet adsorbs magnetic beads dispersed in the liquid drops; then controlling the liquid drop to move continuously through the electrode, and separating the liquid drop from the magnetic beads; the cam continues to rotate, lowering the magnet, releasing the magnetic beads which can be carried away by subsequent droplets, thereby achieving magnetic separation.

Example 2

1. The cell suspension, lysate and reverse transcription reagent are first added to the reservoir region 2 and pre-energized in the corresponding reservoir region.

Formula of lysate reaction solution

Reverse transcription reaction solution formula

2. The electrode driving circuit is controlled by the controller 300 and the circuit control module 200, and the on-off control of the electrodes is performed according to a preset sequence, so that a cell suspension liquid drop is generated from the electrode unit in the liquid storage region 2, and the liquid drop is moved to the area of the hydrophilic site 124A by controlling the driving electrode array connected with the liquid drop. This process is repeated to load each hydrophilic site region with a droplet of cell suspension.

3. By applying a voltage to adjacent electrodes, the position of the cell in the droplet is adjusted so that a single cell is located above the hydrophilic site. And powering off the driving electrode at the hydrophilic point, and waiting for 10s to allow the cells to settle under the action of gravity.

4. Control electrode drive circuit, adjacent to the hydrophilic siteThe cell suspension droplets are passed over the hydrophilic sites by the application of a voltage, which leaves droplets on the hydrophilic sites under the influence of surface tension. After standing, the cells settled under gravity. Applying electricity to the adjacent electrode of the hydrophilic site to move the cell suspension to the adjacent electrode, and controlling the cell concentration of the cell suspension to 10 ⁵ Below one/mL, a small droplet containing a single cell is left on the hydrophilic site at this time. The whole single cell separation process is completed within 30 s. This step can be repeated by continued mixing and splitting of the primary and small droplets until the target cell is obtained. The method is repeated so that each hydrophilic site area is loaded with droplets containing single cells. Single cell isolation is shown, for example, in FIG. 5.

In addition, according to different requirements on the actual liquid drop volume, the uniformity of the generated liquid drop volume is tested, and the result (shown in fig. 6) shows that the liquid drop generation is very stable, and the volume error caused by manual operation is avoided. In addition, the DMF chip can allow the reaction reagent to be added into the liquid storage tank in advance, and all the reagent and the reaction liquid drops are in the silicone oil covering environment to isolate the outside, so that the external pollution can be reduced.

5. The electrode drive circuit is controlled to move the droplets of the primary cell suspension into the waste zone 15 for removal.

6. And controlling an electrode driving circuit to carry out the on-off electric control electrode unit system of the electrodes according to a preset sequence, so that a lysate liquid drop is generated from the liquid storage area 2 and moves to a hydrophilic point area to be mixed with a small liquid drop containing a single cell. This process is repeated so that each hydrophilic site area is loaded with a lysate droplet and mixed with a droplet containing a single cell.

The chip in the prior art is difficult to realize active liquid mixing, so that the detection accuracy problem caused by uneven mixing of reaction reagents is caused. The invention further tests the mixing efficiency of the droplets, and as shown in fig. 7, the two droplets can reach more than 95% of the mixing efficiency of the droplets within 16s, so that the detection of the single-cell transcriptome is more accurate.

7. The chip is placed on a heating plate, and heated at 72 ℃ for 3min to fully lyse the cells.

8. And controlling the electrode driving circuit to perform on-off control of the electrodes according to a preset sequence, so that a reverse transcription reagent droplet is generated from the liquid storage area 2 and moves to the hydrophilic dot area to be mixed with the droplet containing the cell lysate. This process is repeated so that each hydrophilic site region is loaded with a droplet of reverse transcription reagent and mixed with a droplet containing cell lysate.

9. The chip was placed on a flat PCR instrument with the following temperature settings: 42 ℃ for 90min, 65 ℃ for 15min, and 4 ℃ for storage. Completing the reverse transcription reaction of mRNA of single cell to generate cDNA.

10. PCR amplification reagents were added to stock zone 2. And controlling an electrode driving circuit to perform on-off control of the electrodes according to a preset sequence, so that a PCR amplification reagent droplet is generated from the liquid storage area 2 and moves to a hydrophilic dot area to be mixed with the droplet containing the cDNA. This process is repeated so that each hydrophilic site region is loaded with a droplet of cDNA amplification reagent and mixed with a droplet containing cDNA product.

PCR reaction solution formula

11. The chip was placed on a flat PCR instrument with the following temperature settings: 98 degrees 3min, 20 cycles (98 degrees 30s, 67 degrees 30s, 72 degrees 6min), 72 degrees 5min, 4 degrees storage. The cDNA amplification of the single cells was completed.

PCR reaction conditions

12. Purified magnetic bead reagents (commercially available purified magnetic bead reagents, such as Nanjing Novozam Biotech, Inc., N412-01) are added to the stock solution zone 2. And controlling an electrode driving circuit to perform on-off control on the electrodes according to a preset sequence, so that a purified magnetic bead droplet is generated from the electrode unit in the liquid storage area and moves to the hydrophilic dot area to be mixed with the droplet containing the cDNA amplification product. This process is repeated so that each hydrophilic site region is loaded with a droplet of purified magnetic beads and mixed with a droplet containing cDNA amplification products.

13. After standing for 10min, rotating a cam below the chip to lift the magnet, introducing a magnetic field, controlling an electrode driving circuit, performing on-off control on electrodes according to a preset sequence, moving a supernatant part in the liquid drop to a waste liquid zone groove, removing the supernatant part, keeping a magnetic bead part, and removing the magnetic field.

14. Eluent (ultrapure water) was added to the stock solution zone 2. And controlling an electrode driving circuit to carry out on-off control on the electrodes according to a preset sequence, so that eluent liquid drops are generated from the electrode unit in the liquid storage area 2 and move to the magnetic bead area to be mixed with the magnetic beads. This process is repeated so that each bead region is loaded with a droplet of eluent and mixed with the beads.

15. After standing for 10min, rotating a cam below the chip to lift the magnet, introducing a magnetic field, controlling an electrode driving circuit through a control circuit of the integrated circuit, performing on-off control on the electrodes according to a preset sequence, and moving a supernatant part in the liquid drop to an adjacent electrode.

16. And rotating the cam to move the magnet downwards, removing the magnetic field, controlling an electrode driving circuit, performing power-on and power-off control on the electrodes according to a preset sequence, and moving the magnetic beads to a waste liquid area.

17. The library construction reaction is started (commercially available purified magnetic bead reagents, such as Nanjing Novozam Biotech, Inc., TD 503). The library building reaction comprises a fragmentation reaction, a termination reaction and an amplification reaction. Fragmentation reagents were first added to the stock region. And controlling an electrode driving circuit to perform on-off control on the electrodes according to a preset sequence, so that a fragmented reaction droplet is generated from the liquid storage region electrode unit, moves to the supernatant position in the step 15 and is mixed with the supernatant droplet. This process is repeated to mix each fragmented reagent droplet with each supernatant droplet.

18. The chip is placed on a flat PCR instrument, and the reaction temperature is set as follows: preserving at 55 ℃ for 10min and 4 ℃. At this point, a cDNA fragmentation product is generated.

19. A stop reagent was added to the stock solution zone. And controlling an electrode driving circuit to perform on-off control of the electrodes according to a preset sequence, so that a reaction termination droplet is generated from the liquid storage region electrode unit and moved to and mixed with a droplet position containing a cDNA fragmentation product. This process is repeated to mix each of the terminating reagent droplets with each of the cDNA-containing fragmentation products. Standing for 5 min.

20. Amplification reaction reagents were added to the reservoir zone. The control circuit of the integrated circuit controls the electrode driving circuit, and the on-off control of the electrodes is carried out according to a preset sequence, so that a fragmentation reaction liquid drop is generated from the liquid storage region electrode unit, moved to the liquid drop position of the previous step and mixed with the fragmentation reaction liquid drop. This process is repeated to mix each droplet of amplification reagent with each droplet of the previous step.

21. The chip was placed on a flat plate PCR instrument and the reaction temperature was set as follows.

PCR reaction conditions

And (5) repeating the steps of 12-15 to purify the product, and finally obtaining the single-cell transcriptome library product. The product was taken off the chip and directly subjected to the second generation sequencing of illumina.

Claims

1. A single cell transcriptome sequencing library construction platform is characterized by comprising a digital microfluidic chip, a circuit control module, a controller and a magnetic separation control module;

the magnetic separation control module comprises a magnet positioned at the lower part of the digital microfluidic chip and a cam positioned at the bottom of the magnet and used for adjusting the magnet to ascend and descend;

the digital microfluidic chip comprises an upper polar plate and a lower polar plate which are oppositely arranged in parallel, and the upper polar plate and the lower polar plate are separated by a space for liquid movement; the lower electrode plate sequentially comprises a substrate, an electrode layer, a dielectric layer and a hydrophobic layer from bottom to top, and the upper electrode plate sequentially comprises the hydrophobic layer, a grounding conductive layer and a top plate from bottom to top;

the hydrophobic layer comprises at least one hydrophilic spot located at the droplet drive zone to form a micropore;

the plurality of electrode access ports are further connected to a digital microfluidic control instrument, and the control instrument drives liquid drops in the chip to move by applying electric potential between a top plate and a bottom plate of the digital microfluidic chip.

2. The build platform of claim 1, wherein the magnet is a permanent magnet, an electromagnet.

3. A build platform according to claim 1 or 2, wherein the ratio of the magnet diameter to the electrode maximum diameter is 0.5-10.

4. The build platform of claim 1 or 2, wherein the magnetic separation control module further comprises a cam at the bottom of the magnet for adjusting the magnet's ascent and descent.

5. The build platform of claim 1 or 2, wherein the droplet driving region of the lower plate further comprises a reaction region and a plurality of droplet generation channels for connecting the reaction region with the reservoir region for separating droplets from the liquid in the reservoir region for subsequent use in the reaction.

6. The build platform of claim 1 or 2, wherein the array of drive electrodes comprises an array of reaction electrodes and an array of droplet generation channel electrodes positioned to correspond to the reaction zones and droplet generation channels; the droplet generation channel electrode array is used for connecting the reaction electrode array with the reservoir electrode array.

7. The build platform of claim 1 or 2, wherein the fluid storage area further comprises a cell suspension fluid storage area, a lysate fluid storage area, a reverse transcription reagent fluid storage area, a PCR amplification reagent fluid storage area, a magnetic bead reagent fluid storage area, and a waste fluid area;

the liquid storage electrode array comprises a cell suspension liquid storage electrode array, a lysate liquid storage electrode array, a reverse transcription reagent liquid storage electrode array, a PCR amplification reagent liquid storage electrode array, a magnetic bead reagent liquid storage electrode array and a waste liquid electrode array at the corresponding position of the liquid storage area.

8. The build platform of claim 1 or 2, wherein each of said electrode arrays is comprised of at least 1 electrode unit;

preferably, each electrode unit is quadrilateral, and the side of the quadrilateral is linear or zigzag;

preferably, two adjacent electrode edges are fitted to each other;

preferably, the sizes of the electrode units in different said electrode arrays are the same, or partly the same, or different.

9. The build platform of claim 1 or 2, wherein the upper and lower plates are filled with an oil phase; preferably, the oil phase is a silicone oil, such as PMS-200.

10. A method of using a single cell transcriptome sequencing library construction platform, comprising:

a. adding a cell suspension, a lysate and a reverse transcription reagent to the liquid storage region, and pre-electrifying in the corresponding liquid storage region;

b. controlling the electrode unit through the controller and the circuit control module, and performing on-off control on the electrodes according to a preset sequence, so that a cell suspension liquid drop is generated from the electrode unit in the liquid storage area, the liquid drop is moved to a hydrophilic site through controlling a driving electrode array connected with the liquid drop, and the process is repeated to enable each hydrophilic site area to load the cell suspension liquid drop;

c. applying voltage to adjacent electrodes, adjusting the position of the cell in the liquid drop to enable a single cell to be positioned above the hydrophilic site, and powering off the driving electrode at the hydrophilic site to wait for the cell to settle under the action of gravity;

d. a control electrode driving circuit for applying a voltage to the electrodes adjacent to the hydrophilic site to cause droplets of the cell suspension to pass through the hydrophilic site to generate droplets containing single cells;

e. controlling an electrode driving circuit to transfer the original cell suspension liquid drops to a waste liquid area for removal;

f. controlling an electrode driving circuit, and performing on-off electric control electrode unit manufacturing of the electrodes according to a preset sequence, so that a lysate liquid drop is generated from a liquid storage area and moved to a hydrophilic site area to be mixed with the small liquid drop containing the single cell, and repeating the process to enable each hydrophilic site area to load the lysate liquid drop and to be mixed with the small liquid drop containing the single cell;

g. placing the chip on a heating sheet, and fully cracking cells;

h. controlling an electrode driving circuit to perform on-off control of the electrodes according to a preset sequence, so that a reverse transcription reagent droplet is generated from the liquid storage region and moves to a hydrophilic dot region to be mixed with a droplet containing a cell lysis product; repeating the process so that each hydrophilic site region carries a droplet of reverse transcription reagent and is mixed with a droplet containing cell lysate;

i. completing the reverse transcription reaction of mRNA of the single cell to generate cDNA;

j. adding PCR amplification reagent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that a PCR amplification reagent droplet is generated from the liquid storage area and moves to a hydrophilic locus area to be mixed with the droplet containing cDNA, and repeating the process to enable each hydrophilic locus area to load the cDNA amplification reagent droplet and be mixed with the droplet containing cDNA products;

k. performing PCR amplification on the single-cell cDNA in the chip;

adding a purified magnetic bead reagent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that a purified magnetic bead liquid drop is generated from an electrode unit of the liquid storage area and moves to a hydrophilic locus area to be mixed with a liquid drop containing a cDNA amplification product; repeating the process to load each hydrophilic site region with a droplet of purified magnetic beads and mix with the droplet containing the cDNA amplification products;

m, after standing, introducing a magnetic field, controlling an electrode driving circuit, performing on-off control on electrodes according to a preset sequence, moving a supernatant part in the liquid drop to a waste liquid area, removing, keeping a magnetic bead part, and removing the magnetic field;

n, adding eluent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that eluent liquid drops are generated from an electrode unit of the liquid storage area and move to magnetic bead areas to be mixed with magnetic beads, and repeating the process to enable the magnetic bead areas to load the eluent liquid drops and to be mixed with the magnetic beads;

after waiting, introducing a magnetic field, controlling an electrode driving circuit, performing on-off control on electrodes according to a preset sequence, and moving a supernatant part in the liquid drop to an adjacent electrode;

removing the magnetic field, controlling an electrode driving circuit, performing power-on and power-off control on electrodes according to a preset sequence, and transferring the magnetic beads to a waste liquid area;

q, adding the fragmentation reaction reagent into the liquid storage region, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that a fragmentation reaction liquid drop is generated from an electrode unit of the liquid storage region, moved to the supernatant position in the step o and mixed with the supernatant liquid drop; repeating the process to mix each fragmented reagent droplet with each supernatant droplet;

carrying out fragmentation reaction on the single-cell cDNA in the chip to generate a cDNA fragmentation product;

adding a termination reaction reagent into the liquid storage region, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that a termination reaction liquid drop is generated from an electrode unit of the liquid storage region, moved to a liquid drop position containing a cDNA fragmentation product and mixed with the liquid drop position, and repeating the process to mix each termination reaction reagent liquid drop with each cDNA fragmentation product and stand still;

t, adding an amplification reaction reagent into the liquid storage area, controlling an electrode driving circuit, and performing on-off control on electrodes according to a preset sequence, so that a fragmentation reaction liquid drop is generated from an electrode unit of the liquid storage area, moved to the position of the liquid drop in the previous step and mixed with the fragmentation reaction liquid drop; repeating this process to mix each droplet of amplification reagent with each droplet of the previous step;

u, carrying out PCR amplification on the cDNA fragmentation products in the chip, and adding a sequencing joint;

v, repeating the m-o step to purify the product, and finally obtaining a single-cell transcriptome library product; the product was taken off the chip and directly subjected to the second generation sequencing of illumina.