CN113025695A - Sequencing method for high-throughput single-cell chromatin accessibility - Google Patents
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Abstract
The invention discloses a sequencing method for high-throughput single-cell chromatin accessibility, which comprises the following steps: after the cell nucleus is incubated and treated by transposase, the cell nucleus after transposition and a cell label are packaged in a water-in-oil reaction liquid drop by a microfluidic chip; then, the water-in-oil reaction liquid drop is subjected to treatment such as UV illumination, so that the labeled primer is released from the deformable microbeads; and incubating and demulsifying the water-in-oil reaction liquid drop, generating a sequencing library, and finally performing sequencing analysis. The sequencing method has the advantages of high cell capture efficiency, high cell flux, high flexibility, low cross contamination, low double-package rate, high sensitivity, low cost and the like, can realize full automation, does not need electric power drive, can be carried in a portable mode, and has wide application prospect.
Description
Technical Field
The invention relates to a gene sequencing method, in particular to a novel high-throughput single-cell chromatin accessibility sequencing method, and belongs to the field of molecular biology.
Background
The single cell chromatin accessibility sequencing technology can detect the heterogeneity of single cells from the aspect of epigenetics, and provides accurate information for the diagnosis and treatment of diseases. Due to the problems of processing flux, cost and the like of the single cell sequencing technology, a lot of large-scale single cell epigenetic research work cannot be carried out. For example, the traditional single cell separation technology is to separate single cells through a capillary tube, and needs manual operation under a microscope, and has low throughput, long time consumption and tedious process. Another common method is to isolate single cells using a flow cytometer, which requires a large amount of samples, requires precise control, is damaging to cells, and has a high requirement for subsequent bank construction.
It is believed that microfluidic technology combined with single cell chromatin sequencing technology can solve these problems well. For example, the instrument throughput has been increased to a point where hundreds of single cells can be analyzed at one time in a fully automated system of C1 single cells, introduced by Fludigm corporation, usa in 2014. The advent of this system has made it possible to study single-cell chromatin accessibility on a large scale. However, the C1 system uses a micro valve to separate single cells, has low throughput, can only process 938 cells at most, and has high cost. The Microwell-split pool system can also realize the separation of single cells, the flux is also low, usually 1000-. Compared with the C1 system and the Microwell-split pool system, the 10X genomics and Biorad systems based on the droplet microfluidic principle have higher flux, and can simultaneously analyze the chromatin accessibility of tens of thousands of cells. In more detail, 10X genomics and Biorad systems use microfluidic chips to wrap labeled microbeads and single cells in one droplet to achieve separation and labeling of single cells. The flux can reach 1 ten thousand cells. However, the droplet generating devices adopted by the two technologies need to be driven by electric power, so that the cost is high, only 4 or 8 samples can be simultaneously made in each experiment, and the flexibility is limited.
Disclosure of Invention
The main object of the present invention is to provide a sequencing method for high throughput single cell chromatin accessibility, thereby overcoming the disadvantages of the prior art.
In order to achieve the aim of the invention, the invention adopts the following scheme:
the embodiment of the invention provides a sequencing method for high-throughput single-cell chromatin accessibility, which comprises the following steps:
treating the nuclei with a transposase by incubation, such that the open areas of chromatin therein bear the first adaptor sequence, thereby obtaining transposed nuclei;
packing the transposed cell nucleus and the cell label in a water-in-oil reaction liquid drop by using a microfluidic chip, wherein the water-in-oil reaction liquid drop comprises a cell nucleus liquid phase and an oil phase wrapping the cell nucleus liquid phase, the cell nucleus liquid phase comprises a mononuclear cell nucleus and a single cell label, and the cell label comprises deformable microbeads and a labeled primer connected to the deformable microbeads;
physically and/or chemically treating the water-in-oil reaction droplet to release the tagged primers from the deformable beads therein;
incubating the water-in-oil reaction droplet to allow the tagged primer to capture DNA having a first linker sequence therein;
performing demulsification treatment on the water-in-oil reaction liquid drop, extracting and amplifying DNA in the water-in-oil reaction liquid drop, adding sequencing joints at two ends of the DNA to construct a sequencing library, and then performing sequencing analysis.
In some embodiments, the sequencing method comprises: the cells are lysed to obtain nuclei, and the nuclei are incubated with transposase such that the open chromatin region of each nucleus bears a first adaptor sequence which is capable of specifically binding to a second adaptor sequence in the tagged primer, thereby allowing the tagged primer to capture DNA bearing the first adaptor sequence.
In some embodiments, the sequencing method specifically comprises:
preparing the transposed cell nucleus and the cell label into a cell nucleus suspension and a cell label suspension respectively;
providing a micro-fluidic chip which comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port;
respectively injecting the cell nucleus suspension and the cell label suspension into the microfluidic chip, and mixing the cell nucleus suspension and the cell label suspension to form cell nucleus carrier liquid after the cell nucleus suspension and the cell label suspension respectively flow through the cell microchannel and the cell label microchannel;
injecting oil serving as a cell isolation medium into the microfluidic chip, enabling the cell isolation medium to be in contact with the cell nucleus carrier liquid when flowing in the cell isolation medium microchannel, and shearing and wrapping the cell nucleus carrier liquid to form water-in-oil reaction liquid drops containing single cell nuclei and single cell labels;
and collecting the water-in-oil reaction droplets from the single cell sample collection port.
In some embodiments, the sequencing method comprises: by sequencing analysis, at least the position of the chromatin open regions and the positions of the nucleosomes are determined.
The embodiment of the invention also provides a method for constructing a sequencing library with high throughput single cell chromatin accessibility, which comprises the following steps:
incubating and treating the cell nucleus by using transposase to obtain a transposed cell nucleus;
packing the transposed cell nucleus and the cell label in a water-in-oil reaction liquid drop by using a microfluidic chip, wherein the water-in-oil reaction liquid drop comprises a cell nucleus liquid phase and an oil phase wrapping the cell nucleus liquid phase, the cell nucleus liquid phase comprises a mononuclear cell nucleus and a single cell label, and the cell label comprises deformable microbeads and a labeled primer connected to the deformable microbeads;
physically and/or chemically treating the water-in-oil reaction droplet to release the tagged primers from the deformable beads therein;
incubating the water-in-oil reaction droplet such that the tagged primer captures DNA having a first adaptor sequence complementary to a second adaptor sequence using the second adaptor sequence;
incubating the water-in-oil reaction liquid drop, connecting DNA in the water-in-oil reaction liquid drop, and repairing a gap;
and performing demulsification treatment on the water-in-oil reaction liquid drop, extracting and amplifying DNA in the water-in-oil reaction liquid drop, and adding sequencing joints at two ends of the DNA to construct a sequencing library.
The embodiment of the invention also provides a kit for constructing the sequencing library, which comprises:
a microfluidic chip for at least capturing single cell nuclei and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprising an oil phase and a cellular liquid phase encapsulated by the oil phase, and the water-in-oil reaction droplet comprising single cell nuclei and single cell labels;
an oil for forming the oil phase;
a cell lysis reagent;
a cell tag comprising a deformable microbead and a tagged primer attached to the deformable microbead, the tagged primer capable of detaching from the deformable microbead under physical and/or chemical action; and
transposases, nucleic acid amplification reagents, sequencing adaptors, and the like.
In some embodiments, the microfluidic chip comprises a cell microchannel, a cell isolation medium microchannel, a cell label microchannel, and a single-cell nuclear sample collection port, the cell microchannel has a cell nucleus suspension inlet and a single-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 intersects the cell label suspension outlet, so that the single-cell nuclear suspension output by the cell microchannel can be mixed with the cell label suspension output by the cell label microchannel to form a cell nucleus carrier liquid, and the flow path of the cell nucleus carrier liquid intersects the cell isolation medium microchannel, so that the cell isolation medium flowing in the cell isolation medium microchannel can shear and wrap the cell nucleus carrier liquid, thereby forming a water-in-oil reaction droplet containing single cell nucleus and single-cell label, the water-in-oil reaction droplets are output from the single cell nuclear sample collection port.
According to the invention, deformable beads such as hydrogel beads are adopted to construct a cell label, the tail end repair and the completion are carried out in the water-in-oil reaction liquid drop, and an air pump and the like are used as a power source to drive the generation of the water-in-oil reaction liquid drop, so that a plurality of samples can be simultaneously made, the capture efficiency of cells is effectively improved, the mutual pollution of the beads after the liquid drop demulsification is reduced, the proportion of effective data is improved, the cost is reduced, the use mode is more flexible, the operation is easier, and the overall operation time is greatly reduced compared with that of the existing indep and hydropseq platform.
In summary, compared with the prior art, the high-throughput single-cell chromatin accessibility sequencing method provided by the invention has the advantages of high cell capture efficiency, high cell throughput, high flexibility (1-8 samples can be simultaneously made), low cross contamination, low double-wrap rate, high sensitivity (the condition of chromatin open area of a single cell can be detected), low cost and the like, can realize full automation, can detect trace samples, does not need electric power drive, can be carried in a portable way, and has wide application prospect.
Drawings
FIG. 1 is a flow chart of a sequencing process for high throughput single-cell chromatin accessibility in an exemplary embodiment of the invention;
FIG. 2 is a schematic diagram of a microfluidic chip according to an exemplary embodiment of the present invention;
FIG. 3 is a schematic diagram of a water-in-oil reaction droplet generated in a microfluidic chip according to an exemplary embodiment of the present invention;
FIG. 4 is an optical photograph of a water-in-oil reaction droplet generated in an embodiment of the present invention;
FIG. 5 is a Labchip detection profile of DNA extraction according to an embodiment of the present invention;
FIG. 6 is a Labchip detection profile of a sequencing library according to an embodiment of the present invention;
FIG. 7 shows the distribution of the positions of nucleosomes in an embodiment of the invention;
FIG. 8 shows the signal intensity of chromatin opening regions in an embodiment of the invention;
FIG. 9 shows open areas of chromatin on different chromosomes according to an embodiment of the invention.
Detailed Description
One aspect of the embodiments of the present invention provides a sequencing method for high throughput single cell chromatin accessibility.
In summary, the sequencing method comprises a step of cell lysis, a step of transposing cell nuclei, a step of packaging the cell nuclei in a water-in-oil micro-reaction system (water-in-oil reaction droplets), a step of extracting DNA therein for amplification and adding sequencing adapters to form a sequencing library, and a step of sequencing analysis.
Further, the sequencing method may comprise:
treating the nuclei with a transposase by incubation, such that the open areas of chromatin therein bear the first adaptor sequence, thereby obtaining transposed nuclei;
packing the transposed cell nucleus and the cell label in a water-in-oil reaction liquid drop by using a microfluidic chip, wherein the water-in-oil reaction liquid drop comprises a cell nucleus liquid phase and an oil phase wrapping the cell nucleus liquid phase, the cell nucleus liquid phase comprises a mononuclear cell nucleus and a single cell label, and the cell label comprises deformable microbeads and a labeled primer connected to the deformable microbeads;
physically and/or chemically treating the water-in-oil reaction droplet to release the tagged primers from the deformable beads therein;
incubating the water-in-oil reaction droplet to allow the tagged primer to capture DNA having a first linker sequence therein;
performing demulsification treatment on the water-in-oil reaction liquid drop, extracting and amplifying DNA in the water-in-oil reaction liquid drop, adding sequencing joints at two ends of the DNA to construct a sequencing library, and then performing sequencing analysis.
In some embodiments, the sequencing method comprises: the cells are lysed to obtain nuclei, and the nuclei are incubated with transposase such that the open chromatin region of each nucleus bears a first adaptor sequence which is capable of specifically binding to a second adaptor sequence in the tagged primer, thereby allowing the tagged primer to capture DNA bearing the first adaptor sequence.
Further, the transposase includes Tn5 transposase.
In some embodiments, the sequencing method further comprises: after physical and/or chemical treatment of the water-in-oil reaction droplets, incubation is performed at 72-55 ℃ so that the tagged primers capture DNA with the first linker sequence.
More preferably, the collected water-in-oil reaction droplets are subjected to UV irradiation, so that the tagged primers (with the second linker sequence) on the microbeads are freed and bound to the DNA with the first linker sequence.
And incubating the collected water-in-oil reaction droplets at 72 ℃ to 55 ℃ so that the tagged primer can be tightly linked to the DNA with the first adaptor sequence.
Furthermore, the collected water-in-oil reaction droplets can be incubated to link DNA therein and repair gaps.
In some embodiments, the first linker sequence and the second linker sequence are set forth in SEQ ID NO 1 and SEQ ID NO 2, respectively.
In the previous embodiment of the invention, a connection mode is adopted, so that the sequences of the tagged primers on the microbeads can be combined and connected with the DNA with the specific adapter sequences after transposition, the used system is simple, the incubation temperature is low, and the water-in-oil stability is higher. Compared with the prior art (for example, a sequencing technology based on a 10X platform) needs to carry out amplification in a droplet to make a target fragment carry a tag sequence, and the required incubation temperature is high, the requirement on the stability of the droplet is high, and the operation difficulty is high.
The demulsification treatment in the invention preferably adopts physical demulsification modes such as ultrasound and the like, so as to avoid the influence of chemical components such as PFO existing in the chemical demulsification mode on subsequent reactions.
In some embodiments, the sequencing method specifically comprises:
preparing the transposed cell nucleus and the cell label into a cell nucleus suspension and a cell label suspension respectively;
providing a micro-fluidic chip which comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port;
respectively injecting the cell nucleus suspension and the cell label suspension into the microfluidic chip, and mixing the cell nucleus suspension and the cell label suspension to form cell nucleus carrier liquid after the cell nucleus suspension and the cell label suspension respectively flow through the cell microchannel and the cell label microchannel;
injecting oil serving as a cell isolation medium into the microfluidic chip, enabling the cell isolation medium to be in contact with the cell nucleus carrier liquid when flowing in the cell isolation medium microchannel, and shearing and wrapping the cell nucleus carrier liquid to form water-in-oil reaction liquid drops containing single cell nuclei and single cell labels;
and collecting the water-in-oil reaction droplets from the single cell sample collection port.
Further, the cell micro-channel is provided with a cell nucleus suspension inlet and a single cell nucleus suspension outlet, the cell isolation medium micro-channel is provided with a cell label suspension inlet and a cell label suspension outlet, the single cell nucleus suspension outlet and the cell label suspension outlet are intersected, the single cell nucleus suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell nucleus carrier liquid, the flow path of the cell nucleus carrier liquid is intersected with the cell isolation medium micro-channel, so that the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell nucleus carrier liquid into discrete liquid droplet-shaped cell nucleus liquid phase and enable each cell nucleus liquid phase to comprise a single cell nucleus and a single cell label, and the cell isolation medium wraps the cell nucleus liquid phase to form water-in-oil reaction liquid droplets, the water-in-oil reaction droplets are output from the single cell sample collection port.
Of course, a transition section for the cell nucleus carrier liquid to flow may also be provided between the intersection of the single cell nucleus suspension outlet and the cell label suspension outlet and the cell isolation medium micro-channel, which may be named as a cell nucleus carrier liquid micro-channel (as shown by reference numeral 13 in fig. 2), and the cell nucleus carrier liquid micro-channel intersects with the cell isolation medium micro-channel.
Further, the water-in-oil reaction droplets act as water-in-oil microreactors, which can be pico-liter in size.
Furthermore, the microfluidic chip also comprises a cell suspension sample adding cup, a cell isolation medium sample adding cup, a cell label sample adding cup and the like which are respectively communicated with the cell micro-channel, the cell isolation medium micro-channel and the cell label micro-channel.
In some embodiments, a negative pressure motive force generation device is disposed at the single-cell nuclear sample collection port. The negative pressure power generation device can adopt an air pump and the like, and can generate negative pressure in the microfluidic chip so as to drive fluid in each micro-channel to flow. For example, air can be pumped out by an air pump at the single cell nuclear sample collecting port, and negative pressure of-4K to-10K Pa can be applied to the whole microfluidic chip. Through adopting this kind of negative pressure mode, and set up the chip and be 3 passageways, need not drive with power, compare malleation drive among the prior art, multichannel (more than 4 passageways), it is more convenient to operate, and the time also shortens greatly, can do trace sample, and the flexibility is higher, can do 1 to 8 samples simultaneously.
Further, the structure of the microfluidic chip in the foregoing embodiment can be seen from fig. 2, and includes a cell suspension sample cup 1, a cell label sample cup 2, and a cell isolation medium sample cup 4, where the cell suspension sample cup 1, the cell label sample cup 2, and the cell isolation medium sample cup 4 are respectively communicated with a cell micro-channel 11, a cell label micro-channel 12, and a cell isolation medium micro-channel 14, and the microfluidic chip is further provided with a mononuclear cell nuclear sample collecting port 3. Wherein, the cell micro-flow channel 11 and the cell label micro-flow channel 12 are crossed with each other and then crossed with the cell isolation medium micro-flow channel 14, and further communicated with the single cell nuclear sample collecting port 3.
In the above embodiment of the present invention, the cell microchannel of the microfluidic chip is a flow channel for a cell nucleus suspension that is one component for forming a cell nucleus liquid phase, the cell label channel is a flow channel for a cell label suspension that is the other component for forming a cell nucleus liquid phase, the cell isolation medium microchannel is a flow channel for a component that is an oil phase, all components flow at a certain speed along with the flow channel thereof under the condition that pressure is added on the chip, and the cell nucleus suspension liquid and the cell label suspension liquid are mixed to form cell nucleus carrier liquid which is cut by a cell isolation medium as an oil phase to form physical isolation, through controlling the pressure and flow resistance design, the cell isolation medium cuts the single cell nucleus and the cell label, the separation of the single cell and the deformable microbead is realized, and each water-in-oil reaction liquid drop is ensured to be used as a micro-reaction system and comprises a cell and a cell label. For more intuition, the process of forming water-in-oil reaction droplets can also refer to fig. 3, wherein a, b, c, d show the flow directions of the cell label suspension, the cell nucleus liquid phase, and the cell isolation medium, respectively, and e shows the water-in-oil reaction droplets.
In some embodiments, the cell tag comprises a tagged primer and a deformable microbead, the tagged primer is attached to the deformable microbead, and the tagged primer is capable of detaching from the deformable microbead by physical and/or chemical action.
Further, the cell tag recognizes the cell with the tagged primer.
Further, the physical and chemical actions include various physical and chemical actions known to those skilled in the art, such as ultraviolet light irradiation or specific enzyme digestion, etc., and are not limited thereto. For example, the tagged primer may be labeled as an oligonucleotide chain that is uv-sensitive, light-sensitive, or specifically cleaved by an enzyme, and is not limited thereto. In the invention, the method of ultraviolet illumination and the like is preferably adopted to separate the labeled primer from the deformable microbead, which is not only very convenient, but also can not introduce other chemical substances into a micro-reaction system, thereby avoiding potential pollution risk.
Further, the tag includes a base sequence having a length of 5 to 24nt as a barcode.
Further, the barcode may comprise 3 constant base sequences but is not limited to 3.
Further, the total length of the tagged primers may vary from 50nt to 200nt, and is not limited thereto.
In some embodiments, the tagged primer may be chemically and/or physically attached to the deformable microbead. For example, the primer and the bead may be linked by a covalent bond, chemical polymerization, antigen-antibody binding, enzyme-catalyzed linking reaction, and the like, without being limited thereto.
In some embodiments, the deformable beads may be of an organic material or an inorganic-organic composite material, and may be, for example, polyacrylamide gel beads, agarose coated magnetic beads, silica beads, inert material-made beads, and the like, without being limited thereto. Preferably, the deformable beads can be gel beads, which is beneficial to further improving the efficiency of the microfluidic chip for carrying out water-in-oil reaction droplet packaging, and greatly improving the cell flux in cooperation with the microfluidic chip. The mechanism may be as follows: due to the adoption of the deformable beads, the beads with cell labels can be coated in each water-in-oil reaction droplet, the single coating rate can reach 100%, and if the hard beads are replaced, the liquid droplets are coated according to the Poisson distribution rule, so that the actual coating efficiency is far lower than that of the deformable beads. Further, in the present invention, it is preferable to use porous polyacrylamide beads, which carry far more primers than other beads because their specific surface area is much larger than that of other beads, for example, hard beads such as resin beads or magnetic beads. When the polyacrylamide microbeads are synthesized, the concentration of the used acrylamide monomer can be 1% -10%.
In some embodiments, the diameter of the beads may be from 10 μ M to 200 μ M.
In the foregoing embodiment of the invention, with the microfluidic chip, under the condition of the same cell nucleus concentration, the single-port double-inclusion rate is 1/2 with double ports, the cell nuclei with different sizes can be encapsulated by adjusting the pressure (for example, using the pressure generated by the negative pressure power generation device as the power source), especially when the negative pressure power generation device is used as the power source, the encapsulation of the cell nuclei can be rapidly and efficiently completed, and when the gel beads are used to form the cell labels, the suspension flow rate has impact force, the flow rate is controllable, and the encapsulation rate of more than 90% can be realized.
In some embodiments, the sequencing method further comprises: constructing an amplification reaction system based on the extracted DNA and a sequencing joint (for example, the sequence can be shown in SEQ ID NO:3, but is not limited to the sequence) serving as a primer, carrying out PCR amplification, and then purifying to obtain a sequencing library.
In some embodiments, the sequencing method further comprises: by sequencing analysis, at least the position of the chromatin open regions and the positions of the nucleosomes are determined.
In some embodiments, the sequencing method may further include a step of pre-treating the "sample to be tested" or the "sample to be tested". However, with the method provided by the embodiment of the present invention, the requirement for pre-treatment is low, for example, preliminary enrichment can be performed according to the physical or biological characteristics of the cells, and the obtained sample can be used in the subsequent steps.
In this specification, a "test sample" or "test sample" may be derived from an individual (e.g., human blood, biological tissue, etc.) or may be derived from another source, such as some processed or unprocessed laboratory material. In addition, in the present specification, the detection of a "sample" or "specimen" is not only related to a diagnostic purpose, but may also be related to other non-diagnostic purposes.
In another aspect of the embodiments of the present invention, there is provided a kit for constructing the sequencing library, including:
a microfluidic chip for at least capturing single cell nuclei and packaging to generate a water-in-oil reaction droplet, the water-in-oil reaction droplet comprising an oil phase and a cellular liquid phase encapsulated by the oil phase, and the water-in-oil reaction droplet comprising single cell nuclei and single cell labels;
an oil for forming the oil phase;
a cell lysis reagent;
a cell tag comprising a deformable microbead and a tagged primer attached to the deformable microbead, the tagged primer capable of detaching from the deformable microbead under physical and/or chemical action; and
transposases, nucleic acid amplification reagents, sequencing adaptors, and the like.
In the kit provided in this embodiment, the structure and the working principle of the microfluidic chip are as described above, and are not described herein again.
In the kit provided in this embodiment, the composition of the cell label may be as described above, and is not described herein again.
In some embodiments, the kit further comprises: at least reagents required for purifying any one or more of the extracted DNA, sequencing library, such as magnetic beads, etc., and are not limited thereto.
In the aforementioned embodiments of the present invention, the cell lysis reagent may be selected from the types known to those skilled in the art, and may include any protease and protein denaturing reagent suitable for cell lysis, lysis buffer system, etc. known to those skilled in the art.
In the foregoing embodiments of the present invention, the reagents required for amplifying the extracted DNA may include a sequencing linker as a primer and 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, NH4Cl、NaCl、Tris、MgCl2Betaine, DMSO, water, and the like. For example, the DNA polymerase may be selected from Taq DNA polymerase, hot start Taq polymerase, high fidelity enzyme, and the like.
In the preceding embodiments of the invention, the DNA polymerase may be any thermostable DNA polymerase known to those skilled in the art, for example: LA-Taq, rTaq, Phusion, Deep Vent (exo-), Gold 360, Platinum Taq, KAPA 2G Robust, and the like, without being limited thereto.
In the foregoing embodiments of the present invention, the transposition reagent may comprise transposase, transposase reaction buffer, transposition reaction terminator, and the like, which are well known to those skilled in the art. For example, the transposase may be Tn5 or the like, but is not limited thereto.
In the foregoing embodiments of the invention, the sequencing analysis may also be performed in a manner well known to those skilled in the art, which may include basic analysis, standard analysis, and advanced analysis.
The method adopts deformable microbeads such as hydrogel microbeads to construct a cell label, repairs and replenishes the tail end of the cell label in water-in-oil reaction liquid drops, simultaneously utilizes an air pump and the like as a power source to drive the generation of the water-in-oil reaction liquid drops, has the characteristics of high cell capture efficiency, high cell flux and the like, has high flexibility, can simultaneously prepare 1-8 samples, can effectively reduce the mutual pollution of the microbeads after the liquid drops are demulsified, has low cross contamination and low double-package rate, not only can obviously improve the ratio of effective data, but also obviously improve the sensitivity, can detect the condition of a chromatin open area of a single cell, and also reduces the cost (which is less than one third of a sequencing mode based on platforms such as dispseq and 10X), is easier to operate, can realize full automation, does not need electric power to drive, and can be carried.
The invention is further illustrated by the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Unless otherwise specified, various reagents used in the following examples are well known to those skilled in the art and available from commercial sources and the like. However, the experimental methods in the following examples, in which specific conditions are not specified, are generally performed under conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or under the conditions recommended by the manufacturers.
Unless otherwise specified, reagents and consumables used in the following examples, for example: the microfluidic chip has the functions of separating single cell nuclei and generating water-in-oil reaction droplets; oil required for generating the reaction droplets; carrying hydrogel microbeads with tagged primers; nuclease-free water; taq polymerase reaction solution; PCR amplification reaction buffer solution; a DNA-amplifying enzyme; transposase reaction buffer; a transposase; a transposition reaction stop solution; magnetic beads for double-stranded DNA purification; sequencing adapters for library amplification, and the like, are commercially available. For example, the purified magnetic beads used in the following examples may preferably be Ampure XP beads from Beckman, SPRI beads from Beckman, and the like, and are not limited thereto.
Referring to FIG. 1, the sequencing library construction and sequencing process of the following example may include the following steps:
(1) the treated nuclei were incubated with transposase to allow chromatin opening in each nucleus to take place via linker a.
(2) The method comprises the steps of respectively wrapping the transposed cell nucleus and the hydrogel microbead in a liquid drop by using the hydrogel microbead loaded with the labeled primer and a liquid drop microfluidic technology, and releasing the labeled primer on the hydrogel microbead after UV illumination is carried out on the liquid drop.
(3) The droplets were incubated at 72 deg.C-55 deg.C for half an hour to allow the tagged primers to grasp the DNA through the adapter.
(4) Demulsifying, and extracting DNA by using magnetic beads.
(5) Sequencing adapters were added to both sides of the extracted DNA by PCR.
(6) And performing second-generation sequencing on the constructed library, wherein the sequencing scheme is PE 150.
(7) The information data is interpreted to determine the position of the open area of chromatin and the position of nucleosomes.
The specific implementation of this example is described in more detail below.
1. Preparation of the experiment
Preparing cell lysate, and storing reagents: the prepared cell lysate needs to be placed in a refrigerator at 4 ℃ for sealed storage, and the shelf life is 30 days.
TABLE 1 cell lysate Components
| Composition (I) | Volume (μ L) | Final concentration |
| Tris-HCl(1M,PH7.5) | 0.5 | 10mM |
| NaCl(1M) | 0.5 | 10mM |
| MgCl2(0.3M) | 0.5 | 3mM |
| Igepal CA-630(10％) | 0.5 | 0.01％ |
| Nuclease-free water | 48 | / |
| Total | 50 | / |
2. Experimental operation and result display
2.1 cell preparation
2.1.1
Taking a proper amount of PBS solution to put into a water bath kettle for preheating half an hour in advance according to the requirement of the number of processed samples, and taking a proper amount of PBS solution to put into ice for precooling half an hour in advance;
2.1.2
for freshly cultured H1975: digesting with pancreatin to obtain single cells, and centrifuging at normal temperature of 300g for 5 min.
2.1.3
Precooling the centrifuge to 4 ℃, discarding the cell supernatant after centrifugation in 2.1.2, adding 1000 mu L of PBS preheated at 37 ℃ for resuspending the cells, counting, subpackaging 60000 cells (the specific number is determined according to the requirements of a customer) into a 1.5mL centrifuge tube, centrifuging at 4 ℃ for 5min by 500g, discarding the supernatant, and keeping the centrifuge tube filled with cell sediment on ice for placement.
2.1.4
To the 2.1.3 cell pellet was added 50. mu.L of precooled PBS to resuspend the cells, centrifuged at 500g at 4 ℃ for 5min, the supernatant was discarded, and the centrifuge tube containing the cell pellet was kept on ice.
2.1.5
Subpackaging the cell lysate prepared in the step 1 according to the sample size requirement, placing on ice for precooling, adding 50 mu L of precooled cell lysate into 2.1.4 cell sediment, lightly blowing and beating the cell lysate for 20 times by using a pipettor, uniformly mixing, centrifuging at the temperature of 4 ℃ for 10min by 500g, discarding the supernatant, keeping the centrifuge tube with the sediment placed on ice, and immediately carrying out transposition reaction on the sediment.
2.2 transposition reaction
2.2.1 transposition reaction system preparation:
| composition (I) | Volume (μ L) |
| 5× |
10 |
| |
2 |
| Nuclease-free water | 38 |
| Total | 50 |
2.2.2 adding 50 μ L of transposition reaction system in 7.2.1 into 7.1.5 pellet to resuspend cells, gently blowing and beating with a pipette 20 times, and mixing uniformly, wherein the cell pellet is kept on ice in the process;
2.2.3 placing the mixed solution in a metal bath at 37 ℃ for transposition reaction for 30min, and setting the rotating speed of the metal bath to be 300rpm for oscillation;
2.2.4 to the mixture after completion of the transposition reaction of 7.2.3, 12.5. mu.L of a 0.1% SDS solution was added, and the mixture was incubated at room temperature for 5min to terminate the transposition reaction.
By this transposase treatment step, a first linker sequence (defined as linker A) can be attached to its chromatin opening region. For example, the resulting DNA may be one in which the single underlined portion is the linker A and the double underlined portion is the sequence on the transposase.
2.3 Single cell Nuclear encapsulation
2.3.1DNA Capture System formulation
| Reagent | Volume (μ L) |
| Post-transposition nuclear samples | 18 |
| Nuclease-free water | 29.5 |
| Total | 50 |
2.3.2 generating Water-in-oil reaction droplets on a computer (hereinafter referred to simply as droplets)
The transposed cell nucleus, hydrogel and oil were added to the chip shown in FIG. 2, and the volume was pulled from 20ml to 30ml by an air pump (the droplet generation process can refer to FIG. 3), and the size and morphology of the formed droplets can refer to FIG. 4.
Wherein the cell signature can be as follows:
where the single underlined section is the second linker sequence (defined as linker B), the double underlined section is the conventional sequencing primer sequence, and the section containing J, N is the tag sequence, which may be a random sequence.
2.3.4 ultraviolet radiation
UV irradiation is carried out for 10 minutes at the collection port, so that the labeled primers on the microbeads are released, and the transposed DNA is captured.
2.3.3 capturing DNA to repair gaps
The temperature control settings at the collection tube were set as follows:
| step (ii) of | Temperature of | Time |
| The gap is filled up | 72℃ | 5min |
| Annealing trapping | 60 ℃ to 50 ℃ (setting cooling rate 0.3 ℃/s) | 30min |
2.3.4 demulsification
Adding 3ml of PFO into the collecting pipe, blowing and beating for 15 times by using a gun head, centrifuging (800g, 10min, 4 ℃), and taking supernatant;
2.4 extraction of DNA
Taking out Ampure XP beads half an hour in advance, balancing to room temperature, and purifying the PCR product by using Ampure XP beads through magnetic beads for 1.2 Xbeads; labchip quality inspection was performed as shown in FIG. 5.
2.5 library construction
2.5.1 Add Joint
| Reagent | Volume (μ L) |
| Extracted DNA sample | 18 |
| ATAC-I7(10uM) | 2.5 |
| N50X | 2.5 |
| 2×KAPA HIFI |
25 |
| Nuclease- |
2 |
| Total | 50 |
Wherein, the sequence of ATAC-I7 is shown in SEQ ID NO. 3.
2.6 purification of the product
2.6.1 taking out Ampure XP beads half an hour ahead of time and balancing to room temperature, and purifying the PCR product by using Ampure XP beads through magnetic beads (0.8X + 0.7X for purification);
2.6.2 checking the total volume of the product in the PCR tube, and adding water to make up to 50 mu L;
2.6.3 adding 40 μ L Ampure XP beads (0.8 x) into the PCR tube, gently blowing, mixing, and incubating at room temperature for 5 min;
2.6.4 transferring the PCR tube to a magnetic frame for standing for 2min, and transferring the supernatant to a new PCR tube after the solution is clarified;
2.6.5 Add 35. mu.L of Ampure XP beads (0.7X) to the new PCR tube solution, gently blow and mix well, incubate for 5min at room temperature;
2.6.6 placing the PCR tube in 2.5.5 on a magnetic frame and standing for 2min, and discarding the supernatant containing small fragments after the solution is clarified;
2.6.7PCR tube is kept on magnetic frame, 150 μ L of freshly prepared and pre-cooled 80% ethanol solution is added, and after standing for 30s, ethanol is discarded;
2.6.8 repeat 2.6.7 once;
2.6.9 keeping the PCR tube on the magnetic frame for 3-5min, and drying the magnetic beads in the air without reflecting light;
2.6.10 adding 20 μ L TE solution into the PCR tube, blowing and mixing uniformly for 20 times, and incubating for 5min at room temperature;
2.6.11 placing the PCR tube on a magnetic frame, standing for 2min, transferring the supernatant to a new PCR tube after the solution is clarified, and taking care not to wash the magnetic beads in the step;
2.6.12 mu.L of the Qubit dsDNA High Sensitivity Assay was used for concentration determination, 1. mu.L of the sample was sent to Labchip quality inspection, and the library size was around 200-800bp, as shown in FIG. 6.
3. Sequencing analysis
The sequencing process adopts a PE150 sequencing scheme, can be carried out on Illumina Nova Seq, Illumina Hiseq, Illumina Nextseq 500, Illumina Miseq and other platforms, and corresponding operation methods and experimental conditions are well known to those skilled in the art. Corresponding sequencing analysis can be seen in FIGS. 7-9.
Referring to FIG. 7, the double-ended ATAC-Seq sequencing reads can embody nucleosome packaging and localization information. Sequencing reads showed a periodic distribution of approximately 200bp in insert length. The ATAC-Seq signal intensity of the 3kb region upstream and downstream of the Transcription Start Site (TSS) is shown in FIG. 8. And fig. 9 shows the number and distribution of peak on the chromosome.
In addition, the inventors of the present application also utilized the same nuclear samples as in this example, based on some existing sequencing protocols, such as: a typical high quality conventional ontology ATAC-seq scheme; cusanovich et al, Science, 5 months and 22 days 2015; 348(6237) 910-14; buenrostro et al, Nature, 23/7/2015; 523(7561) 486-90; ideal sequencing measurements for ATAC-seq experiments, control experiments were performed.
Comparing the data obtained from these control experiments with the data obtained from the examples of the present invention, it is verified that the sequencing method provided by the examples of the present invention is ideal in terms of cell throughput, accuracy, sensitivity, etc.
All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.
Sequence listing
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Claims (12)
1. A method for sequencing chromatin accessibility to a high throughput single cell, comprising:
treating the nuclei with a transposase by incubation, such that the open areas of chromatin therein bear the first adaptor sequence, thereby obtaining transposed nuclei;
packing the transposed cell nucleus and the cell label in a water-in-oil reaction liquid drop by using a microfluidic chip, wherein the water-in-oil reaction liquid drop comprises a cell nucleus liquid phase and an oil phase wrapping the cell nucleus liquid phase, the cell nucleus liquid phase comprises a mononuclear cell nucleus and a single cell label, and the cell label comprises deformable microbeads and a labeled primer connected to the deformable microbeads;
physically and/or chemically treating the water-in-oil reaction droplet to release the tagged primers from the deformable beads therein;
incubating the water-in-oil reaction droplet to allow the tagged primer to capture DNA having a first linker sequence therein;
performing demulsification treatment on the water-in-oil reaction liquid drop, extracting and amplifying DNA in the water-in-oil reaction liquid drop, adding sequencing joints at two ends of the DNA to construct a sequencing library, and then performing sequencing analysis.
2. The sequencing method of claim 1, comprising: the cells are lysed to obtain nuclei, and the nuclei are incubated with transposase such that the open chromatin region of each nucleus bears a first adaptor sequence which is capable of specifically binding to a second adaptor sequence in the tagged primer, thereby allowing the tagged primer to capture DNA bearing the first adaptor sequence.
3. The sequencing method of claim 1, comprising: demulsifying the water-in-oil reaction liquid drop by adopting a physical mode; preferably, the physical means comprises sonication means.
4. The sequencing method of claim 2, further comprising:
after the water-in-oil reaction liquid drop is subjected to physical and/or chemical treatment, the incubation is carried out at 72-55 ℃, so that the DNA with the first adaptor sequence is captured by the tagged primer; and
and incubating the water-in-oil reaction liquid drop, connecting DNA in the water-in-oil reaction liquid drop, and repairing gaps.
5. The sequencing method of claim 1, wherein: the first linker sequence and the second linker sequence are respectively shown as SEQ ID NO 1 and SEQ ID NO 2; and/or the total length of the tagged primers is 50nt to 200 nt; and/or, the tag comprises a base sequence of 5-24nt in length as a barcode; preferably, the barcode comprises 3 constant base sequences.
6. The sequencing method of claim 1, wherein: irradiating the water-in-oil reaction droplet with ultraviolet light, thereby causing the tagged primers therein to be released from the deformable beads.
7. The sequencing method of claim 1, wherein: the deformable beads comprise gel beads, preferably porous polyacrylamide beads; and/or the diameter of the deformable microbead is 10-200 μ M.
8. The sequencing method of claim 1, wherein: the transposase includes Tn5 transposase.
9. The sequencing method according to claim 1, characterized in that it comprises in particular:
preparing the transposed cell nucleus and the cell label into a cell nucleus suspension and a cell label suspension respectively;
providing a micro-fluidic chip which comprises a cell micro-channel, a cell isolation medium micro-channel, a cell label micro-channel and a single-cell sample collecting port;
respectively injecting the cell nucleus suspension and the cell label suspension into the microfluidic chip, and mixing the cell nucleus suspension and the cell label suspension to form cell nucleus carrier liquid after the cell nucleus suspension and the cell label suspension respectively flow through the cell microchannel and the cell label microchannel;
injecting oil serving as a cell isolation medium into the microfluidic chip, enabling the cell isolation medium to be in contact with the cell nucleus carrier liquid when flowing in the cell isolation medium microchannel, and shearing and wrapping the cell nucleus carrier liquid to form water-in-oil reaction liquid drops containing single cell nuclei and single cell labels;
and collecting the water-in-oil reaction droplets from the single cell sample collection port.
10. The sequencing method of claim 9, wherein: the cell micro-channel is provided with a cell nucleus suspension inlet and a single cell nucleus suspension outlet, the cell isolation medium micro-channel is provided with a cell label suspension inlet and a cell label suspension outlet, the single cell nucleus suspension outlet and the cell label suspension outlet are intersected, so that the single cell nucleus suspension output by the cell micro-channel can be mixed with the cell label suspension output by the cell label micro-channel to form cell nucleus carrier liquid, the flow path of the cell nucleus carrier liquid is crossed with the cell isolation medium micro-channel, the cell isolation medium flowing through the cell isolation medium micro-channel can shear the continuous cell nucleus carrier liquid into discrete liquid droplet-shaped cell nucleus liquid phase, each cell nucleus liquid phase comprises a single cell nucleus and a single cell label, and the cell isolation medium wraps the cell nucleus liquid phase to form water-in-oil reaction liquid droplets, the water-in-oil reaction droplets are output from the single cell sample collection port.
11. The sequencing method according to claim 9 or 10, characterized in that: and a negative pressure power generation device arranged at the single cell nuclear sample collection port is utilized to generate negative pressure in the microfluidic chip, so that the fluid in each micro-channel is driven to flow.
12. The sequencing method of claim 1, comprising: and constructing an amplification reaction system based on the extracted DNA and a sequencing joint serving as a primer for PCR amplification, and then purifying to obtain a sequencing library.
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| CN113604545A (en) * | 2021-08-09 | 2021-11-05 | 浙江大学 | Ultrahigh-throughput single-cell chromatin transposase accessibility sequencing method |
| CN114471761A (en) * | 2022-02-15 | 2022-05-13 | 天津诺威百奥科技有限公司 | Can dismantle chip liquid drop and generate appearance |
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| CN113604545A (en) * | 2021-08-09 | 2021-11-05 | 浙江大学 | Ultrahigh-throughput single-cell chromatin transposase accessibility sequencing method |
| CN113604545B (en) * | 2021-08-09 | 2022-04-29 | 浙江大学 | Ultrahigh-throughput single-cell chromatin transposase accessibility sequencing method |
| CN114471761A (en) * | 2022-02-15 | 2022-05-13 | 天津诺威百奥科技有限公司 | Can dismantle chip liquid drop and generate appearance |
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