WO2021128034A1 - High-throughput sequencing method for single-cell chromatins accessibility - Google Patents

Abstract

A high-throughput sequencing method for single-cell chromatins accessibility, comprising: incubation treating a cell nucleus by using a transposase, and then packaging the cell nucleus subjected to transposition and a cell tag into a water-in-oil reaction droplet by using a microfluidic chip; subjecting the water-in-oil reaction droplet to treatment such as UV irradiation, so that a primer with a tag therein is released from deformable beads; and incubating and demulsifying the water-in-oil reaction droplet, then generating a sequencing library, and finally performing sequencing analysis. Said sequencing method has the advantages of high cell capturing efficiency, high cell throughput, high flexibility, low cross-contamination, low double packaging rate, high sensitivity, and low cost, and can be fully automated, does not need to be driven by electricity, and is portable, providing a wide application prospect.

Description

High-throughput single-cell chromatin accessibility sequencing method Technical field

This application relates to a gene sequencing method, in particular to a novel high-throughput single-cell chromatin accessibility sequencing method, which belongs to the field of molecular biology.

Background technique

Single-cell chromatin accessibility sequencing technology can detect the heterogeneity of single cells from the aspect of epigenetics, and provide accurate information for the diagnosis and treatment of diseases. Constrained by the throughput and cost of single-cell sequencing technology, many large-scale single-cell epigenetics research work cannot be carried out. For example, the traditional single-cell separation technology uses capillary tubes to separate single cells, which requires manual operation under a microscope, which has low throughput, time-consuming, and cumbersome process. Another common method is to use flow cytometry to separate single cells. This method requires a large amount of samples, requires precise control, damages the cells, and requires high requirements for subsequent library building.

The industry believes that the combination of microfluidic technology and single-cell chromatin sequencing technology can solve these problems well. For example, the C1 single cell fully automated system launched by Fludigm in the United States in 2014 has increased the throughput of this instrument to the ability to analyze hundreds of single cells at a time. The emergence of this system makes it possible to study the accessibility of single-cell chromatin on a large scale. However, this type of C1 system uses microvalves to separate single cells, and the throughput is low. Currently, it can only produce 938 cells at most, and the cost is high. The Microwell-split pool system can also realize the separation of single cells, and its throughput is also low, usually 1000-5000 cells, the reaction volume is large, the reagent consumption is high, the cost is high, and the cell loss is large. Compared with the C1 system and the Microwell-split pool system, the 10X genomics and Biorad systems based on the principle of droplet microfluidics have higher throughput and can analyze the chromatin accessibility of tens of thousands of cells at the same time. In more detail, the 10X genomics and Biorad systems use microfluidic chips to wrap labeled beads and single cells in a droplet to achieve the separation and labeling of single cells. The flux can reach 10,000 cells. However, the droplet generating devices used in these two technologies need to be driven by electricity, and the cost is high. Each experiment can only do 4 or 8 samples at the same time, and the flexibility is limited.

Summary of the invention

The main purpose of this application is to provide a high-throughput single-cell chromatin accessibility sequencing method, so as to overcome the shortcomings of the prior art.

In order to achieve the foregoing invention objectives, this application adopts the following solutions:

The embodiment of the present application provides a high-throughput single-cell chromatin accessibility sequencing method, which includes:

Incubate the cell nucleus with transposase to make the chromatin open region with the first linker sequence to obtain the transposed cell nucleus;

A microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet. The water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase. The phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;

Physical and/or chemical treatment of the water-in-oil reaction droplets so that the labeled primers are released from the deformable microbeads;

Incubating the water-in-oil reaction droplet so that the labeled primer captures the DNA with the first linker sequence therein;

The water-in-oil reaction droplets are demulsified, the DNA is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library, and then sequencing analysis is performed.

In some embodiments, the sequencing method includes: lysing the cell to obtain a nucleus, and then incubating the nucleus with a transposase, so that the chromatin open zone of each nucleus is provided with a first linker sequence, and the first linker sequence It can specifically bind to the second linker sequence in the tagged primer, so that the tagged primer can capture the DNA with the first linker sequence.

In some embodiments, the sequencing method specifically includes:

The cell nucleus and cell label after transposition are respectively made into a cell nucleus suspension and a cell label suspension;

Provide a microfluidic chip, which includes a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single cell sample collection port;

Inject the cell nucleus suspension and the cell label suspension into the microfluidic chip respectively, and make the cell nucleus suspension and the cell label suspension flow through the cell microchannel and the cell label microchannel respectively and then mix with each other to form a nuclear carrier liquid;

The oil as the cell isolation medium is injected into the microfluidic chip, and the cell isolation medium is brought into contact with the nucleus carrier fluid when flowing in the cell isolation medium microchannel, and the nucleus carrier fluid is sheared and wrapped to form a single cell nucleus and Single cell labelled water-in-oil reaction droplets;

And, collecting the water-in-oil reaction droplets from the single-cell sample collection port.

In some embodiments, the sequencing method includes: at least determining the position of the open chromatin region and the position of the nucleosome through sequencing analysis.

The embodiments of the present application also provide a method for constructing a sequencing library with high-throughput single-cell chromatin accessibility, which includes:

Incubate the cell nucleus with transposase to obtain the nucleus after transposition;

A microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet. The water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase. The phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;

Physical and/or chemical treatment of the water-in-oil reaction droplets so that the labeled primers are released from the deformable microbeads;

Incubating the water-in-oil reaction droplet, so that the tagged primer captures the DNA with the first linker sequence by using the second linker sequence it carries, and the first linker sequence is complementary to the second linker sequence;

Incubating the water-in-oil reaction droplet, connecting the DNA therein, and repairing the gap;

Demulsification treatment is performed on the water-in-oil reaction droplets, and the DNA therein is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library.

The embodiment of the present application also provides a kit for constructing the sequencing library, which includes:

The microfluidic chip is used at least to capture and package single cell nuclei to generate water-in-oil reaction droplets. The water-in-oil reaction droplets include an oil phase and a cell liquid phase wrapped by the oil phase, and the oil The water reaction droplet contains single cell nucleus and single cell label;

Oil used to form the oil phase;

Cell lysis reagent;

The cell label includes a deformable microbead and a labeled primer attached to the deformable microbead, and the labeled primer can be separated from the deformable microbead under physical and/or chemical action; and

Transposase, nucleic acid amplification reagents, sequencing adapters, etc.

In some embodiments, the microfluidic chip includes a cell microfluidic channel, a cell isolation medium microfluidic channel, a cell label microfluidic channel, and a single-cell nuclear sample collection port, and the cell microfluidic channel has a cell nuclear suspension inlet and a single cell nucleus sample collection port. The cell nucleus suspension outlet, the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet, and the single-cell nucleus suspension outlet and the cell label suspension outlet meet to make the cell microchannel The output single cell nuclear suspension can be mixed with the cell label suspension output from the cell label microchannel to form a nucleus carrier liquid. The flow path of the cell nucleus carrier liquid crosses the cell isolation medium microchannel, so that The cell isolation medium flowing in the microchannel of the cell isolation medium can shear and wrap the nucleus carrier fluid, thereby forming a water-in-oil reaction droplet containing a single cell nucleus and a single cell label, and the water-in-oil reaction droplet is composed of a single-cell nucleus sample Collect port output.

In this application, deformable microbeads such as hydrogel microbeads are used to construct cell labels, and terminal repair and supplementation are performed in water-in-oil reaction droplets. At the same time, an air pump is used as a power source to drive the formation of water-in-oil reaction droplets. Multiple samples can be taken at the same time, which effectively improves the capture efficiency of cells, reduces the mutual contamination of microbeads after the droplets are demulsified, increases the proportion of effective data, and reduces the cost, the use is more flexible, and the operation is easier Compared with the existing indrop and dropseq platforms, the overall operation time is greatly reduced.

All in all, compared with the prior art, the high-throughput single-cell chromatin accessibility sequencing method provided by this application has high cell capture efficiency, high cell throughput, and high flexibility (1-8 samples can be made at the same time), Low cross-contamination, low double-packing rate, high sensitivity (detection of the chromatin open area of a single cell), low cost and other advantages, and can be fully automated, can detect small samples without power driving, and can be carried in a portable way. The application prospect is broad.

Description of the drawings

FIG. 1 is a process flow diagram of a high-throughput single-cell chromatin accessibility sequencing process in a typical embodiment of the present application;

FIG. 2 is a schematic structural diagram of a microfluidic chip in an exemplary embodiment of the present application;

3 is a schematic diagram of the generation of water-in-oil reaction droplets in a microfluidic chip in a typical embodiment of the present application;

Fig. 4 is an optical photograph of a water-in-oil reaction droplet generated in a specific implementation case of the present application;

Figure 5 is a Labchip detection map of DNA extracted in a specific implementation case of this application;

Fig. 6 is a Labchip detection map of a sequencing library in a specific implementation case of this application;

Figure 7 shows the location distribution of nucleosomes in a specific implementation case of the present application;

Figure 8 shows the signal intensity of the chromatin open area in a specific implementation case of the present application;

Figure 9 shows the open regions of chromatin on different chromosomes in a specific implementation case of the present application.

Detailed ways

One aspect of the embodiments of the present application provides a high-throughput single-cell chromatin accessibility sequencing method.

In summary, the sequencing method includes the steps of lysing the cells, transposing the nuclei, and packaging the nuclei in a water-in-oil microreaction system (water-in-oil reaction droplets), and extracting the The steps of amplifying DNA and adding sequencing adapters to form a sequencing library, and performing sequencing analysis steps.

Further, the sequencing method may include:

Incubate the cell nucleus with transposase to make the chromatin open region with the first linker sequence to obtain the transposed cell nucleus;

A microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet. The water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase. The phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;

Physical and/or chemical treatment of the water-in-oil reaction droplets so that the labeled primers are released from the deformable microbeads;

Incubating the water-in-oil reaction droplet so that the labeled primer captures the DNA with the first linker sequence therein;

The water-in-oil reaction droplets are demulsified, the DNA is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library, and then sequencing analysis is performed.

In some embodiments, the sequencing method includes: lysing the cell to obtain a nucleus, and then incubating the nucleus with a transposase, so that the chromatin open zone of each nucleus has a first linker sequence, and the first linker The sequence can specifically bind to the second linker sequence in the tagged primer, so that the tagged primer can capture the DNA with the first linker sequence.

Further, the transposase includes Tn5 transposase.

In some embodiments, the sequencing method further includes: performing physical and/or chemical treatment on the water-in-oil reaction droplet, and then incubating at 72°C-55°C to make the labeled primer capture the band DNA with the first linker sequence.

More preferably, the collected water-in-oil reaction droplets can be irradiated with ultraviolet light to free the labeled primer (with the second linker sequence) on the microbeads and bind to the DNA with the first linker sequence.

And, incubate the collected water-in-oil reaction droplets at 72°C-55°C, so that the tagged primer can be closely linked to the DNA with the first linker sequence.

Furthermore, the collected water-in-oil reaction droplets can also be incubated to connect the DNA therein and repair the gap.

In some embodiments, the first linker sequence and the second linker sequence are shown in SEQ ID NO: 1 and SEQ ID NO: 2, respectively.

In the foregoing embodiments of the present application, a connection method is adopted, so that the tagged primer sequence on the microbead can be combined with and connected to the DNA with a specific linker sequence after transposition. The system used is simple, the incubation temperature is low, and the water-in-oil The stability will be higher. In contrast, the prior art (for example, sequencing technology based on the 10X platform) requires amplification in the droplet to make the target fragment carry the tag sequence. The incubation temperature required is high, and the stability of the droplet is required. High and difficult to operate.

The demulsification treatment in this application preferably adopts physical demulsification methods such as ultrasound to avoid the influence of chemical components such as PFO existing in the chemical demulsification method on subsequent reactions.

In some embodiments, the sequencing method specifically includes:

The cell nucleus and cell label after transposition are respectively made into a cell nucleus suspension and a cell label suspension;

Provide a microfluidic chip, which includes a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single cell sample collection port;

Inject the cell nucleus suspension and the cell label suspension into the microfluidic chip respectively, and make the cell nucleus suspension and the cell label suspension flow through the cell microchannel and the cell label microchannel respectively and then mix with each other to form a nuclear carrier liquid;

The oil as the cell isolation medium is injected into the microfluidic chip, and the cell isolation medium is brought into contact with the nucleus carrier fluid when flowing in the cell isolation medium microchannel, and the nucleus carrier fluid is sheared and wrapped to form a single cell nucleus and Single cell labelled water-in-oil reaction droplets;

And, collecting the water-in-oil reaction droplets from the single-cell sample collection port.

Further, the cell microchannel has a cell nuclear suspension inlet and a single cell nuclear suspension outlet, the cell isolation medium microchannel has a cell label suspension inlet and a cell label suspension outlet, and the single cell nuclear suspension outlet Intersect with the cell label suspension outlet, so that the single cell nucleus suspension output from the cell microchannel can be mixed with the cell label suspension output from the cell label microchannel to form a nucleus carrier liquid, and the flow path of the cell nucleus carrier liquid Intersect the microchannels of the cell isolation medium, so that the cell isolation medium flowing through the microchannels of the cell isolation medium can shear the continuous nuclear carrier fluid into discrete droplet-shaped nuclear liquid phases and make each cell nuclear fluid The phase includes a single cell nucleus and a single cell label, while the cell isolation medium is allowed to wrap the cell liquid phase to form water-in-oil reaction droplets, which are output from the single-cell sample collection port.

Of course, a transition section for the flow of the nucleus carrier liquid can also be provided between the intersection of the single-cell nucleus suspension outlet and the cell label suspension outlet and the microchannel of the cell isolation medium, which can be named nucleus carrier liquid microfluidics. (Shown as mark 13 in Fig. 2), the nucleus-carrying liquid microchannel and the cell isolation medium microchannel intersect.

Further, the water-in-oil reaction droplets are used as water-in-oil microreactors, and their size can be upgraded.

Further, the microfluidic chip also includes a cell suspension sample cup, a cell separation medium sample cup, and a cell label that are respectively connected to the cell microchannel, cell isolation medium microchannel, and cell label microchannel. Refill cups, etc.

In some embodiments, a negative pressure power generation device is provided at the single-cell nuclear sample collection port. The negative pressure power generation device may use an air pump or the like, which can generate negative pressure in the microfluidic chip to drive fluid flow in each microfluidic channel. For example, an air pump can be used to extract air from the single-cell nuclear sample collection port, and a negative pressure of -4K to -10K Pa can be applied to the entire microfluidic chip. By adopting this negative pressure method and setting the chip as 3 channels, it does not need to be driven by power. Compared with the positive pressure drive and multi-channel (above 4 channels) in the prior art, the operation is more convenient, the time is greatly shortened, and the It is more flexible to do micro samples, and you can do 1 to 8 samples at the same time.

Further, the structure of a microfluidic chip in the foregoing embodiment can be referred to as shown in Figure 2, including a cell suspension sample cup 1, a cell label sample cup 2, a cell isolation medium sample cup 4, and the cell suspension The liquid sample addition cup 1, the cell label sample cup 2, the cell isolation medium sample cup 4 are respectively connected with the cell microchannel 11, the cell label microchannel 12, and the cell isolation medium microchannel 14, and the microfluidic control The chip is also provided with a single-cell nuclear sample collection port 3. Wherein, after the cell microchannel 11 and the cell label microchannel 12 intersect each other, they also cross the cell isolation medium microchannel 14 and further communicate with the single-cell nuclear sample collection port 3.

In the above embodiments of the present application, the cell microfluidic channel of the microfluidic chip is a flow channel for forming a nuclear suspension of one component of the cell nucleus liquid phase, and the cell labeling channel is another flow channel for forming the nuclear liquid phase. The flow channel of the cell label suspension of the components, the cell isolation medium micro flow channel is the flow channel of the oil phase components. All the components flow at a certain speed along with the flow channel when the pressure on the chip is increased. And the nucleus carrier liquid formed by mixing the cell nucleus suspension and the cell label suspension is cut by the cell isolation medium as the oil phase to form a physical isolation. By controlling the pressure and flow resistance design, the cell isolation medium can cut a single cell nucleus and cell label. , To achieve the separation of single cells and deformable beads, to ensure that each water-in-oil reaction droplet serves as a micro-reaction system and contains a cell and a cell label. More intuitively, the process of forming water-in-oil reaction droplets can also refer to Figure 3, where a, b, c, and d respectively show the cell label suspension, cell nuclear suspension, cell nuclear liquid phase, and cell isolation medium. The flow direction, e shows the water-in-oil reaction droplets.

In some embodiments, the cell label includes a labeled primer and a deformable bead, the labeled primer is attached to the deformable bead, and the labeled primer can be physically and/or chemically Detach from deformable microbeads under the action.

Further, the cell tag uses the labeled primer to identify the cell.

Further, the physical and chemical effects include various physical and chemical effects well known to those skilled in the art. For example, ultraviolet light irradiation or specific enzyme cleavage can be preferably used, and it is not limited thereto. For example, the labeled primer may be labeled as an oligonucleotide chain that is ultraviolet-sensitive, light-sensitive, or can be specifically cleaved, and is not limited thereto. In this application, it is preferable to use ultraviolet light to remove the labeled primer from the deformable beads, which is not only very convenient, but also does not introduce other chemical substances into the microreaction system, thereby avoiding potential pollution risks. In addition, The free labeled primer captures the target product more efficiently, and can eliminate the problem of low capture efficiency caused by the steric hindrance effect of the microbead when the primer fixed on the microbead captures the target product.

Further, the tag includes a base sequence of 5-24 nt as a barcode.

Furthermore, the barcode may include 3 but not limited to 3 constant base sequences.

Furthermore, the total length of the tagged primers may range from 50 nt to 200 nt, and is not limited thereto.

In some embodiments, the labeled primer may be chemically and/or physically attached to the deformable microbead. For example, the primer and the microbead can be connected by covalent bonding, chemical polymerization, antigen-antibody binding, enzyme-catalyzed connection reaction, etc., and it is not limited thereto.

In some embodiments, the deformable beads can be made of organic materials or inorganic-organic composite materials, for example, polyacrylamide gel beads, agarose gel beads, agarose-coated magnetic beads, Silicon beads, microbeads made of inert materials, etc. are not limited thereto. Preferably, the deformable microbeads can be gel microbeads, which are beneficial to further improve the efficiency of the microfluidic chip for water-in-oil reaction droplet packaging, and cooperate with the microfluidic chip to greatly increase cell flux . The mechanism may be: due to the use of deformable microbeads, each water-in-oil reaction droplet can be coated with cell-labeled microbeads, and the single packaging rate can reach 100%. If it is replaced with hard microbeads, When the droplets are wrapped, the Poisson distribution rules must be followed, and the actual wrapping efficiency is much lower than that of deformable beads. Furthermore, porous polyacrylamide microbeads are preferably used in this application, because their specific surface area is much larger than that of other microbeads, such as hard microbeads such as resin or magnetic microbeads, so the number of primers carried is far more than that of hard microbeads. Other microbeads. When synthesizing the polyacrylamide microbeads, the concentration of acrylamide monomer used can be 1%-10%.

In some embodiments, the diameter of the microbeads may be 10 μM-200 μM.

In the foregoing embodiment of the present application, using the microfluidic chip, the single-port double-packing rate is 1/2 of the double-port under the same cell nucleus concentration, and cell nuclei of different sizes can be adjusted by pressure (for example, with negative The pressure generated by the pressure force generating device is used as the power) to realize the wrapping, especially when the negative pressure power generating device is used as the power source, the nucleus wrapping can be completed quickly and efficiently, and the gel beads are used to form the cell label. The suspension flow rate has an impact force, the flow rate is controllable, and the wrapping rate can be over 90%.

In some embodiments, the sequencing method further includes: constructing an amplification reaction system based on the extracted DNA and a sequencing adapter as a primer (for example, the sequence may be shown in SEQ ID NO: 3, but is not limited to this). After PCR amplification, purification is performed to obtain a sequencing library.

In some embodiments, the sequencing method further includes: at least determining the position of the open chromatin region and the position of the nucleosome through sequencing analysis.

In some embodiments, the sequencing method may further include a step of pre-processing the "sample to be tested" or the "sample to be tested". However, applying the methods provided in the embodiments of the present application requires relatively low pre-processing. For example, preliminary enrichment can be performed according to the physical or biological characteristics of the cells, and the obtained samples can be used in subsequent steps.

In this specification, "sample to be tested" or "sample to be tested" can come from an individual (such as human blood, biological tissue, etc.), or from other sources, such as some processed or unprocessed laboratory materials . In addition, in this specification, the detection of "sample" or "sample" does not only involve diagnostic purposes, but may also involve other non-diagnostic purposes.

Another aspect of the embodiments of the present application also provides a kit, which is used to construct the sequencing library, and includes:

The microfluidic chip is used at least to capture and package single cell nuclei to generate water-in-oil reaction droplets. The water-in-oil reaction droplets include an oil phase and a cell liquid phase wrapped by the oil phase. The water reaction droplet contains single cell nucleus and single cell label;

Oil used to form the oil phase;

Cell lysis reagent;

The cell label includes a deformable microbead and a labeled primer attached to the deformable microbead, and the labeled primer can be separated from the deformable microbead under physical and/or chemical action; and

Transposase, nucleic acid amplification reagents, sequencing adapters, etc.

In the kit provided in this embodiment, the structure and working principle of the microfluidic chip are as described above, and will not be repeated here.

In the kit provided in this embodiment, the composition of the cell label can be as described above, and will not be repeated here.

In some embodiments, the kit further includes: at least the reagents required to purify any one or more of the extracted DNA and the sequencing library, such as magnetic beads, etc., and are not limited thereto.

In the foregoing embodiments of the present application, the cell lysis reagent used can be of a type well known to those skilled in the art, and it can include any protease and protein denaturing reagents and lysis buffers that are well known to those skilled in the art that are suitable for cell lysis. System and so on.

In the foregoing embodiments of the present application, the reagents required for amplifying the extracted DNA may include a sequencing adapter as a primer, a DNA polymerase and an amplification buffer well-known to those skilled in the art. For example, the main components of the amplification buffer may include: KCl, NH ₄ Cl, NaCl, Tris, MgCl ₂ , betaine, DMSO, water, and the like. For example, the DNA polymerase can be selected from Taq DNA polymerase, hot-start Taq polymerase, high-fidelity enzymes and the like.

In the foregoing embodiments of the present application, the DNA polymerase can be any thermostable DNA polymerase known to those skilled in the art, such as: LA-Taq, rTaq, Phusion, Deep Vent, Deep Vent (exo-) , Gold 360, Platinum Taq, KAPA 2G Robust, etc., but not limited to this.

In the foregoing embodiments of the present application, the transposition reagent may include a transposase, a transposase reaction buffer, a transposition reaction stop solution, etc., which are well known to those skilled in the art. For example, the transposase can be selected from Tn5 and the like, and is not limited thereto.

In the foregoing embodiments of the present application, the sequencing analysis can also be performed in a manner well known to those skilled in the art, which can include basic analysis, standard analysis, and advanced analysis.

In this application, deformable microbeads such as hydrogel microbeads are used to construct cell labels, and terminal repair and supplementation are performed in water-in-oil reaction droplets. At the same time, an air pump is used as a power source to drive the formation of water-in-oil reaction droplets. It has the characteristics of high cell capture efficiency, high cell throughput, and high flexibility. It can do 1-8 samples at the same time, and can effectively reduce the mutual contamination of microbeads after droplet demulsification, low cross-contamination, and low double-packing rate. Not only can significantly increase the proportion of valid data, but also significantly improve the sensitivity, detect the chromatin open area of a single cell, and reduce the cost (one third of the sequencing methods based on dropseq, 10X, etc.) Below), the operation is easier, can be fully automated, does not require electric drive, and can be portable.

The application will be further elaborated below in conjunction with specific embodiments. It should be understood that these embodiments are only used to illustrate the application and not to limit the scope of the application. Unless otherwise specified, the various reagents used in the following examples are well known to those skilled in the art and can be obtained through market purchases and other channels. However, the experimental methods that do not specify specific conditions in the following examples are usually based on conventional conditions such as those described in J. Sambrook et al., Molecular Cloning Experiment Guide, Third Edition, Science Press, 2002, or according to the conditions described in manufacturing The conditions suggested by the manufacturer.

Unless otherwise specified, the reagents and consumables used in the following examples, for example: a microfluidic chip with the functions of single-cell nucleus separation and water-in-oil reaction droplet generation; the oil required to generate the reaction droplets; carrying a label Primer hydrogel beads; nuclease-free water; Taq polymerase reaction solution; PCR amplification reaction buffer; DNA amplification enzyme; transposase reaction buffer; transposase; transposition reaction termination solution; for Magnetic beads for double-stranded DNA purification; sequencing adapters for library amplification, etc., are all commercially available. For example, the purified magnetic beads used in the following embodiments may preferably be Ampure XP beads of Beckman Company, SPRI beads of Beckman Company, etc., and are not limited thereto.

Referring to FIG. 1, the sequencing library construction and sequencing process of the following embodiment may include the following steps:

(1) Incubate the processed cell nuclei with transposase so that the chromatin open area of each cell nucleus is covered with a joint A.

(2) Utilize the hydrogel microbeads and droplet microfluidic technology with labeled primers to wrap the transposed cell nucleus and the hydrogel microbeads in the droplets respectively, and apply UV light to the droplets. The labeled primers on the hydrogel beads are released.

(3) Incubate the droplet at 72°C-55°C for half an hour, so that the labeled primer can grab the DNA through the adapter.

(4) Demulsification, using magnetic beads to extract DNA.

(5) Add sequencing adapters on both sides of the extracted DNA by PCR.

(6) Perform second-generation sequencing on the established library, and the sequencing scheme is PE150.

(7) Interpretation of information data to determine the location of the open area of chromatin and the location of nucleosomes.

The specific implementation process of this embodiment will be described in more detail below.

1. Experiment preparation

Preparation of cell lysate, reagent storage requirements: The prepared cell lysate must be stored in a refrigerator at 4°C and sealed, with a shelf life of 30 days.

Table 1 Cell lysate components

ingredient	Volume (μL)	Final concentration
Tris-HCl(1M, PH7.5)	0.5	10mM
NaCl(1M)	0.5	10mM
MgCl 2 (0.3M)	0.5	3mM
Igepal CA-630 (10%)	0.5	0.01%
Nuclease-free water	48	/
Total	50	/

2. Experimental operation and results display

2.1 Cell preparation

2.1.1

According to the needs of the number of samples to be processed, take an appropriate amount of PBS solution and put it in a water bath half an hour in advance to preheat, and take an appropriate amount of PBS solution half an hour in advance and put it on ice to pre-cool;

2.1.2

For freshly cultured H1975: trypsin digestion to obtain single cells, centrifuge at 300g at room temperature for 5 minutes.

2.1.3

Pre-cool the centrifuge to 4 ℃, discard the cell supernatant after centrifugation in 2.1.2, add 1000 μL of 37 ℃ pre-warmed PBS to resuspend the cells, and count, aliquot 60,000 cells (the specific number is based on the customer (Determined by the demand) in a 1.5mL centrifuge tube, centrifuge at 500g at 4°C for 5min, discard the supernatant, and keep the centrifuge tube containing the cell pellet on ice.

2.1.4

Add 50μL of pre-cooled PBS to the cell pellet in 2.1.3 to resuspend the cells, centrifuge at 500g at 4°C for 5 minutes, discard the supernatant, and keep the centrifuge tube containing the cell pellet on ice.

2.1.5

Divide the cell lysate prepared in 1 according to the sample volume, and place it on ice to pre-cool. Add 50μL of the pre-cooled cell lysate to the 2.1.4 cell pellet, and gently pipette 20 times to mix. Centrifuge at 500g for 10 min at 4°C, discard the supernatant, and keep the centrifuge tube with the sediment on ice. The sediment will immediately undergo transposition reaction.

2.2 Transposition reaction

2.2.1 Preparation of transposition reaction system:

ingredient	Volume (μL)
5×TAG Buffer	10
Tn5 transposase	2
Nuclease-free water	38
Total	50

2.2.2 Add the 50μL transposition reaction system in 7.2.1 to the pellet in 7.1.5 to resuspend the cells, gently pipette 20 times to mix, and the cell pellet must be kept on ice during this process;

2.2.3 Place the above mixed solution in a metal bath at 37°C for a transposition reaction for 30 minutes, and set the metal bath rotation speed to 300 rpm for shaking;

2.2.4 Add 12.5 μL of 0.1% SDS solution to the mixture after the transposition reaction of 7.2.3 is completed, and incubate at room temperature for 5 minutes to terminate the transposition reaction.

Through this step of transposase treatment, the first linker sequence (defined as linker A) can be placed in the open zone of chromatin. For example, a kind of DNA obtained can be as shown below, wherein the part marked with a single underline is the linker A, and the part marked with a double underline is the sequence on the transposase.

2.3 Single-cell nuclear package

2.3.1 DNA capture system preparation

Reagent

Volume (μL)

Nuclear sample after transposition	18
Nuclease-free water	29.5
Total	50

2.3.2 On the machine to generate water-in-oil reaction droplets (hereinafter referred to as droplets)

Add the transposed cell nucleus, hydrogel, and oil to the chip shown in Figure 2, and press the air pump to pull the volume from 20ml to 30ml (see Figure 3 for the droplet generation process), and the resulting droplet size and morphology Refer to Figure 4.

The cell label can be as follows:

The part marked with a single underline is the second linker sequence (defined as linker B), the part marked with a double underline is the sequence of a conventional sequencing primer, and the part containing J and N is a tag sequence, which may be a random sequence.

2.3.4 UV irradiation

The collection port was irradiated with ultraviolet for 10 minutes to release the labeled primers on the beads and capture the transposed DNA.

2.3.3 Capture DNA repair gaps

Set the temperature control setting at the collection tube as follows:

step	temperature	time
Fill in the gap	72°C	5min
Annealing trap	60°C—50°C (set cooling rate 0.3°C/s)	30min

2.3.4 Demulsification

Add 3ml PFO to the collection tube, pipette 15 times with a pipette tip, centrifuge (800g, 10min, 4℃), and take the supernatant;

2.4 Extract DNA

Half an hour in advance, Ampure XP beads were taken out to equilibrate to room temperature, and the above PCR products were purified by magnetic beads using Ampure XP beads to purify 1.2X beads; Labchip quality inspection observation, as shown in Figure 5.

2.5 Library construction

2.5.1 Add connector

Reagent	Volume (μL)
DNA sample after extraction	18

ATAC-I7(10uM)	2.5
N50X	2.5
2×KAPA HIFI Hotstart Ready Mix	25
Nuclease-free water	2
Total	50

Among them, the sequence of ATAC-I7 is shown in SEQ ID NO: 3.

2.6 Product purification

2.6.1 Take out Ampure XP beads half an hour in advance to equilibrate to room temperature, and use Ampure XP beads to purify the above PCR products with magnetic beads (0.8×+0.7× for purification);

2.6.2 Check the total volume of the product in the PCR tube, add water to make up to 50μL;

2.6.3 Add 40μL Ampure XP beads (0.8×) to the above PCR tube, gently pipette to mix, and incubate at room temperature for 5 minutes;

2.6.4 Transfer the above PCR tube to a magnetic stand and let it stand for 2 minutes. After the solution is clear, transfer the supernatant to a new PCR tube;

2.6.5 Add 35μL Ampure XP beads (0.7×) to the solution of the new PCR tube, gently pipette to mix, and incubate at room temperature for 5 minutes;

2.6.6 Place the PCR tube in 2.5.5 on a magnetic stand and let it stand for 2 minutes. After the solution is clear, discard the supernatant containing small fragments;

2.6.7 Keep the PCR tube on the magnetic stand, add 150μL of freshly prepared and pre-cooled 80% ethanol solution to it, and discard the ethanol after standing for 30s;

2.6.8 Repeat 2.6.7 once;

2.6.9 Keep the PCR tube on the magnetic rack for 3-5 minutes, and wait for the magnetic beads to dry and not reflect light;

2.6.10 Add 20 μL TE solution to the PCR tube, mix by pipetting 20 times, and incubate at room temperature for 5 minutes;

2.6.11 Place the PCR tube on the magnetic stand and let it stand for 2 minutes. After the solution is clear, transfer the supernatant to a new PCR tube. Be careful not to wash the magnetic beads in this step;

2.6.12 Take 1μL Qubit dsDNA High Sensitivity Assay for concentration determination, take 1μL sample and send it to Labchip quality inspection, the library size is about 200-800bp, as shown in Figure 6.

3. Sequencing analysis

The sequencing process adopts the PE150 sequencing solution, which can be performed on platforms such as Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, and Illumina Miseq. The corresponding operation methods and experimental conditions are well known to those skilled in the art. The corresponding sequencing analysis can be seen in Figure 7-9.

Among them, referring to Figure 7, paired-end ATAC-Seq sequencing reads can reflect the information of nucleosome packaging and positioning. The insert length of the sequencing reads will show a periodic distribution of approximately 200bp. Refer to Figure 8 for the ATAC-Seq signal intensity of the 3kb region upstream and downstream of the transcription start site (TSS). Figure 9 shows the number and distribution of peaks on the chromosome.

In addition, the inventor of this application also uses the same cell nucleus sample as in this example, based on some existing sequencing solutions, such as: a typical high-quality traditional ontology ATAC-seq solution; Cusanovich et al., Science, May 22, 2015 ; 348(6237):910-14; Buenrostro et al., Nature, July 23, 2015; 523(7561):486-90; The ideal sequencing metric for ATAC-seq experiments, a controlled experiment was carried out.

By comparing the data obtained in these control experiments with the data obtained in the examples of this application, it is confirmed that the sequencing methods provided in the examples of this application are ideal in terms of cell throughput, accuracy, and sensitivity.

All documents mentioned in this application are cited as references in this application, as if each document was individually cited as a reference. In addition, it should be understood that after reading the above teaching content of this application, those skilled in the art can make various changes or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims of this application.

Claims (12)

1. A high-throughput single-cell chromatin accessibility sequencing method, which is characterized in that it comprises:

   Incubate the cell nucleus with transposase to make the chromatin open region with the first linker sequence to obtain the transposed cell nucleus;

   A microfluidic chip is used to package the transposed cell nucleus and cell label in a water-in-oil reaction droplet. The water-in-oil reaction droplet includes a nucleus liquid phase and an oil phase that wraps the nucleus liquid phase. The phase includes a single-cell nucleus and a single-cell label, and the cell label includes a deformable microbead and a labeled primer connected to the deformable microbead;

   Physical and/or chemical treatment of the water-in-oil reaction droplets so that the labeled primers are released from the deformable microbeads;

   Incubating the water-in-oil reaction droplet so that the labeled primer captures the DNA with the first linker sequence therein;

   The water-in-oil reaction droplets are demulsified, the DNA is extracted and amplified, and sequencing adapters are added at both ends of the DNA to construct a sequencing library, and then sequencing analysis is performed.
2. The sequencing method according to claim 1, characterized in that it comprises: lysing the cell to obtain a nucleus, and then incubating the nucleus with a transposase, so that the open chromatin zone of each nucleus is provided with a first linker sequence, A linker sequence can specifically bind to the second linker sequence in the tagged primer, so that the tagged primer can capture the DNA with the first linker sequence.
3. The sequencing method according to claim 1, characterized in that it comprises: using a physical method to demulsify the water-in-oil reaction droplets; preferably, the physical method includes an ultrasonic treatment method.
4. The sequencing method according to claim 2, characterized in that it further comprises:

   After the water-in-oil reaction droplets are physically and/or chemically treated, they are then incubated at 72°C-55°C, so that the tagged primers capture the DNA with the first linker sequence; and

   Incubate the water-in-oil reaction droplet, connect the DNA therein, and repair the gap.
5. The sequencing method according to claim 1, wherein the first linker sequence and the second linker sequence are as shown in SEQ ID NO: 1 and SEQ ID NO: 2 respectively; and/or, the tagged The total length of the primers is 50 nt-200 nt; and/or, the tag includes a base sequence of 5-24 nt as a barcode; preferably, the barcode includes three constant base sequences.
6. The sequencing method according to claim 1, wherein the water-in-oil reaction droplets are irradiated with ultraviolet light, so that the labeled primers are released from the deformable beads.
7. The sequencing method according to claim 1, wherein the deformable beads comprise gel beads, preferably porous polyacrylamide beads; and/or, the diameter of the deformable beads is 10 μM- 200μM.
8. The sequencing method according to claim 1, wherein the transposase comprises Tn5 transposase.
9. The sequencing method according to claim 1, characterized in that it specifically comprises:

   The cell nucleus and cell label after transposition are respectively made into a cell nucleus suspension and a cell label suspension;

   Provide a microfluidic chip, which includes a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single cell sample collection port;

   Inject the cell nucleus suspension and the cell label suspension into the microfluidic chip respectively, and make the cell nucleus suspension and the cell label suspension flow through the cell microchannel and the cell label microchannel respectively and then mix with each other to form a nuclear carrier liquid;

   The oil as the cell isolation medium is injected into the microfluidic chip, and the cell isolation medium is brought into contact with the nucleus carrier fluid when flowing in the cell isolation medium microchannel, and the nucleus carrier fluid is sheared and wrapped to form a single cell nucleus and Single cell labelled water-in-oil reaction droplets;

   And, collecting the water-in-oil reaction droplets from the single-cell sample collection port.
10. The sequencing method according to claim 9, wherein the cell microfluidic channel has a nuclear suspension inlet and a single-cell nuclear suspension outlet, and the cell isolation medium microfluidic channel has a cell label suspension inlet and a cell label suspension. Liquid outlet, and the single-cell nucleus suspension outlet and the cell label suspension outlet meet, so that the single-cell nucleus suspension output from the cell microchannel can be mixed with the cell label suspension output from the cell label microchannel to form a cell nucleus A carrier fluid, the flow path of the cell nucleus carrier fluid intersects with the cell isolation medium microchannels, so that the cell isolation medium flowing through the cell isolation medium microchannels can shear the continuous nucleus carrier fluid into discrete droplets The cell nucleus liquid phase and each nucleus liquid phase contain single cell nucleus and single cell label, and the cell isolation medium is allowed to wrap the cell liquid phase to form water-in-oil reaction droplets. The reaction droplets are output from the single-cell sample collection port.
11. The sequencing method according to claim 9 or 10, characterized in that: a negative pressure power generation device arranged at the single-cell nuclear sample collection port is used to generate negative pressure in the microfluidic chip, thereby driving the fluid in each microfluidic channel flow.
12. The sequencing method according to claim 1, characterized by comprising: constructing an amplification reaction system based on the extracted DNA and sequencing adapters as primers, performing PCR amplification, and then purifying to obtain a sequencing library.

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