Single-cell sequencing based on droplet microfluidics and its applications

Abstract

The present invention provides a sequencing library and an application thereof. The provided sequencing library comprises: a first nucleic acid molecule, which carries a cell tag sequence and a droplet tag sequence; and a second nucleic acid molecule, which carries an insert fragment and a cell tag sequence.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to single-cell sequencing based on droplet microfluidics and its application. Background Art

[0002] Single-cell sequencing technology is one of the most popular technologies in the past decade, and has shown a wide range of application prospects in development, tumor-related scientific research and disease diagnosis. Currently, single-cell sequencing technology has been achieved at different levels such as genome, transcriptome, epigenome and proteome, and has played an important role in the research of many scientific problems. However, single-cell sequencing technology currently faces many challenges in terms of data quality, throughput, cost, operability, etc., and there are still many technical barriers to its real large-scale application.

[0003] Omic sequencing technology based on microfluidic systems is one of the most popular areas in recent years. Compared with traditional technologies, the microscale droplet field in microfluidics is characterized by its extremely small reagent consumption, which can greatly reduce the cost of expensive reagent consumption. Each droplet with a stable morphology can be regarded as an independent microreactor, which reduces cross-contamination and is easy to manipulate. Its large specific surface area can also accelerate various reaction rates and heat transfer rates. It is an ideal choice for single-cell research. Compared with other microfluidic technologies, droplet microfluidics has gradually become a popular means of biochemical analysis. However, at present, most single-cell library construction and sequencing technologies based on droplet microfluidic platforms require the input of hundreds of thousands of cells, but can only recover a few thousand single cells, resulting in a large amount of cell sample waste. Reducing the amount of cell input and improving cell recovery rate through the invention and use of new biochemical strategies is one of the most important research hotspots at present. Summary of the invention

[0004] The present invention aims to solve at least one of the technical problems in the related art to a certain extent. When using droplet microfluidics technology for single-cell sequencing, the waste of cell samples can be avoided by reducing the amount of cell input and improving the recovery rate and utilization rate of cells, which is more suitable for the purpose of sequencing a small number of samples.

[0005] Single-cell sequencing based on droplet microfluidics technology has become an ideal means of single-cell research with small reagent usage and simple manipulation. Referring to the literature published on cell resource (Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets, Evan Z. Macosko et al., Cell 161, 1202-1214, May 21, 2015), droplet microfluidics technology uses oil-in-water droplets to encapsulate cells and micro-particles to obtain droplets containing a cell and a micro-particle at the same time. Other components in the droplet also include lysate, in which the micro-beads contain a large number of specific identical barcodes (cell label sequences, i.e., cellbarcodes, used as markers for reads from the same cell). During the continuous generation of droplets, the cells in the droplets are cracked by the lysate, releasing a large amount of mRNA, and at the same time, the mRNA can be captured by the micro-beads. After all droplets are generated, the droplets are broken. The mRNA is reverse transcribed into cDNA and further amplified. Then, the cDNA is sheared by a shearing enzyme and sequencing adapters are added to both ends. Further, the cDNA with adapters is sequenced.

[0006] However, droplet microfluidics technology also faces a big disadvantage, that is, the initial cell input is too high (usually more than 100,000), and the cells finally obtained only account for a very low proportion of the input cells (about 4%). One of the main reasons is that the distribution of cells and microbeads in the droplets is a random event, which conforms to the Poisson distribution. Single-cell sequencing needs to ensure that the droplet contains only one cell and one microbead, and there cannot be more than 2 different cells or 2 different microbeads in the same droplet. In order to meet this requirement, cells and microbeads need to be prepared at a very low concentration, so that a large number of droplets do not contain cells and microbeads at the same time, or there is only one cell or only one microbead. In the end, a large number of cells are wasted, and the number of cells that meet the sequencing requirements is only about 4% of the number of cells input, which causes a large amount of waste of samples. How to reduce the input of cells and increase the proportion of cells obtained to avoid a large amount of waste of samples, especially suitable for single-cell sequencing of a small number of samples, is the focus of the present invention.

[0007] During the research process, the inventors creatively thought of whether it is possible to allow multiple microbeads to be encapsulated in a droplet, and then distinguish these microbeads from the same droplet to increase the proportion of cells that meet the sequencing requirements. For example, a droplet label sequence can be added to each different droplet as a mark to identify reads from different droplets. There is also a cell label sequence in the droplet as a mark to identify reads from different cells. The sequencing library constructed in this way contains both cell label sequences and droplet label sequences. During sequencing, by identifying these label sequences, different reads can be attributed to different cells, thereby realizing single-cell sequencing. According to an embodiment of the present invention, the surface of the microbeads in the same droplet carries a cell label sequence and also contains a capture sequence. The capture sequence is used to capture the inserted fragments from the cell (i.e., the fragments for which the corresponding sequencing information is to be obtained by sequencing), and the nucleic acid molecules carrying the inserted fragments and the cell label sequence can be obtained in the sequencing library finally formed; at the same time, the inserted fragments from the same cell may be captured by microbeads carrying different cell label sequences, which makes it impossible to effectively distinguish them after demulsification, so the droplet label sequence is added to the droplet at the same time, so that the droplet label sequence can be captured by the capture sequence on the surface of the microbeads in the droplet at the same time, and the nucleic acid molecules carrying the cell label sequence and the droplet label sequence can be obtained in the sequencing library finally formed. Thus, by sequencing the nucleic acid molecules carrying the inserted fragments and the cell label sequence, and the nucleic acid molecules carrying the cell label sequence and the droplet label sequence, the sequencing results are mutually verified to confirm the reads from the same cell, thereby obtaining the single-cell sequencing results.

[0008] In this way, the construction of sequencing libraries and single-cell sequencing does not require only one microsphere to be encapsulated in a droplet, so a very low concentration of microspheres is not required. As a result, more cells will be captured by the microspheres, thereby reducing the amount of sample input. It can be manifested as: the input amount of cell samples is reduced from more than 100,000 to less than 10,000, and the cell recovery rate is also increased from 4% to more than 50%. The available cells obtained in a single experiment are 4000-6000, which can greatly reduce the cost of single-cell library construction and sequencing, which is beneficial to various studies or detections carried out on a large scale through single-cell sequencing. In addition, it is known to those skilled in the art that any other available carrier that can achieve the same function as the microsphere is also included in the scope of protection claimed by the present invention.

[0009] Specifically, the present invention provides the following technical solutions:

[0010] In a first aspect of the present invention, the present invention provides a sequencing library, comprising: a first nucleic acid molecule, the first nucleic acid molecule carrying a cell tag sequence and a droplet tag sequence; and a second nucleic acid molecule, the second nucleic acid molecule carrying an insert and a cell tag sequence. As described above, the present invention confirms reads from the same cell by sequencing nucleic acid molecules carrying inserts and cell tag sequences, and nucleic acid molecules carrying cell tag sequences and droplet tag sequences, and the sequencing results are mutually verified, thereby obtaining single-cell sequencing results.

[0011] According to an embodiment of the present invention, the sequencing library described above may further include the following technical features:

[0012] According to an embodiment of the present invention, the second nucleic acid molecule further includes a molecular tag sequence. The second nucleic acid molecule further carries a molecular tag sequence, which can indicate reads from different genes, thereby achieving accurate determination of different genes in different cells.

[0013] According to an embodiment of the present invention, the molecular tag sequence length is 6 to 15 nt, preferably 8 to 12 nt, and more preferably 10 nt. A molecular tag sequence of 6 to 15 bases in length can form 4 6 ~4 15 For example, the length of the molecular tag sequence can be 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt or 15nt, etc., which can be used to indicate reads from different genes without occupying too much sequencing information and causing unnecessary waste.

[0014] According to an embodiment of the present invention, the insert fragment is formed based on an mRNA molecule or a DNA molecule. The insert fragment carried by the second nucleic acid molecule can be an mRNA molecule after cell lysis, or a DNA molecule. Taking mRNA molecules as an example, mRNA molecules usually carry polyA sequences, so when microbeads or other carriers are used for capture, polyT sequences can be used as corresponding capture sequences to achieve capture of the insert fragment.

[0015] According to an embodiment of the present invention, the cell tag sequence length is 10 to 16 nt. The cell tag sequence of 10 to 16 bases in length can form 4 10 ~4 16 Different sequences can be used to indicate different cells without taking up too much sequencing information and causing unnecessary waste.

[0016] According to an embodiment of the present invention, the length of the droplet tag sequence is 6 to 15 nt, preferably 8 to 12 nt, and more preferably 10 nt. The droplet tag sequence of 6 to 15 bases in length can form 4 6 ~4 15 For example, the length of the droplet label sequence can be 6nt, 7nt, 8nt, 9nt, 10nt, 11nt, 12nt, 13nt, 14nt or 15nt, etc., which can be used to indicate sequencing reads from the same droplet without occupying too much sequencing information and causing unnecessary waste.

[0017] In the second aspect of the present invention, the present invention provides a droplet, the droplet containing: biological material, the biological material containing nucleic acid molecules; droplet identification molecules, the droplet identification molecules carrying droplet label sequences; and a first carrier, the first carrier carrying a capture sequence and a cell label sequence, wherein the capture sequence is used to capture at least one of the nucleic acid molecules and the droplet identification molecules. According to an embodiment of the present invention, the first carrier may further contain an amplification identification sequence. As described above, the present application provides a droplet containing both a droplet identification molecule and a carrier, which can be used to indicate sequencing reads from the same cell, and a droplet contains only one cell, which can reduce the amount of sample used and improve the utilization rate of cells during single-cell sequencing, and is particularly suitable for single-cell sequencing of a small number of samples.

[0018] According to an embodiment of the present invention, the above droplet may further include the following technical features:

[0019] According to an embodiment of the present invention, the nucleic acid molecule is mRNA or DNA.

[0020] According to an embodiment of the present invention, the biological material is provided in the form of cells.

[0021] According to an embodiment of the present invention, each of the droplets contains one of the cells, and each of the droplets contains at least one of the carriers.

[0022] According to an embodiment of the present invention, it further comprises: a cell lysis reagent. The cell lysis reagent can be used to lyse biological materials, such as cells, in the droplets, so as to facilitate the capture sequence on the carrier to capture the nucleic acid molecules on the biological material.

[0023] According to an embodiment of the present invention, the droplet identification molecule further includes a recognition sequence of a capture sequence, and the recognition sequence of the capture sequence is connected to the droplet tag sequence. The droplet identification molecule further includes a recognition sequence of a capture sequence, and the recognition sequence of the capture sequence can be recognized by the capture sequence on the carrier, thereby facilitating the carrier to recognize and capture the droplet identification molecule.

[0024] According to an embodiment of the present invention, the droplet identification molecule is provided in the form of a long-chain molecule, and the long-chain molecule includes: a plurality of the droplet identification molecules connected in series; wherein the droplet label sequence on the same long-chain molecule has the same nucleic acid sequence. The droplet identification molecule is provided in the form of a long-chain molecule, and the long-chain molecule contains a plurality of droplet identification molecules connected in series. By processing the long-chain molecule, for example, an endonuclease recognition sequence can be inserted between the droplet identification molecules, and then the endonuclease is used for enzymatic digestion, so that a plurality of droplet identification molecules can be obtained, which are used to indicate sequencing reads from different droplets in different droplets. Multiple droplet identification molecules can also be obtained by other means, for example, oligonucleic acids with cell label sequences fixed on some beads as the first carrier can be released, combined with long-chain molecules, and then obtained by extension or amplification reaction.

[0025] According to an embodiment of the present invention, the long-chain molecule further includes an endonuclease recognition sequence, the endonuclease recognition sequence is arranged between two adjacent droplet identification molecules, and the droplet further includes an endonuclease, which is suitable for cutting the endonuclease recognition sequence. The droplet also contains an endonuclease, and the endonuclease is used to recognize the endonuclease recognition sequence, so that the long-chain molecule can be processed easily, and a large number of droplet identification molecules can be quickly obtained and applied to different droplets.

[0026] According to an embodiment of the present invention, the endonuclease recognition sequence is a double-stranded sequence, and the droplet identification molecule is a single-stranded sequence.

[0027] According to an embodiment of the present invention, the droplet identification molecule is provided in the form of a second carrier, and the second carrier carries a plurality of droplet identification molecules. According to an embodiment of the present invention, the plurality of droplet identification molecules can be connected to the second carrier by covalent bonds or other means. For example, it can be connected to the carrier by a disulfide bond, which is convenient for the subsequent use of some covalent bond breaking reagents, such as DTT to destroy the covalent bond and release the droplet identification molecules, thereby facilitating the capture of the capture sequence carried on the first carrier. Or the second carrier can be a hydrogel microparticle, and the plurality of droplet identification molecules can be embedded in the hydrogel microparticles, and the hydrogel is melted under appropriate reaction conditions to release the droplet identification molecules. The other methods mentioned can be a connection method similar to a covalent bond, but it will not destroy the hydrogen bond.

[0028] According to an embodiment of the present invention, the droplet identification molecule further includes a recognition sequence of a capture sequence, and the recognition sequence of the capture sequence is connected to the droplet tag sequence, thereby facilitating the capture sequence carried on the first carrier to specifically bind to the recognition sequence of the capture sequence, thereby capturing the droplet tag sequence.

[0029] According to an embodiment of the present invention, the covalent bond is a disulfide bond.

[0030] According to an embodiment of the present invention, the droplet further comprises: a cleavage reagent, which can destroy the connection between the plurality of droplet identification molecules and the second carrier. The cleavage reagent mentioned in the text can be used as long as it can destroy the covalent bond or other modified connection between the droplet identification molecules and the second carrier mentioned above.

[0031] According to an embodiment of the present invention, the cleavage reagent is DTT.

[0032] According to an embodiment of the present invention, the droplet is a water-in-oil structure.

[0033] In a third aspect of the present invention, the present invention provides a suspension comprising a plurality of droplets, wherein the droplets are droplets according to any embodiment of the second aspect of the present invention, wherein for at least two droplets, the droplet label sequences have different nucleic acid sequences. By preparing a suspension comprising a plurality of droplets, the suspensions can be sequenced and analyzed to obtain desired sequencing results.

[0034] In a fourth aspect of the present invention, the present invention provides a cell dispersion, comprising: cells and long-chain molecules, wherein the long-chain molecules comprise: a plurality of series-connected droplet tag sequences; wherein the droplet tag sequences on the same long-chain molecules have the same nucleic acid sequence. The provided cell dispersion can be used with a carrier, such as microbeads, to prepare droplets through a microfluidic chip, and the droplets are demulsified, library built, sequenced, etc., to obtain sequencing results of single cells in the cell dispersion.

[0035] According to an embodiment of the present invention, the cell dispersion liquid described above may further include the following technical features:

[0036] According to an embodiment of the present invention, the cell is in a single cell form.

[0037] According to an embodiment of the present invention, a plurality of long-chain molecules are included, wherein, for at least two long-chain molecules, the droplet tag sequence has different nucleic acid sequences.

[0038] According to an embodiment of the present invention, the long-chain molecule further comprises: an endonuclease recognition sequence, and the endonuclease recognition sequence is arranged between two adjacent droplet tag sequences.

[0039] According to an embodiment of the present invention, the endonuclease recognition sequence is a double-stranded sequence, and the droplet tag sequence is a single-stranded sequence.

[0040] According to an embodiment of the present invention, the long-chain molecule is provided in the form of DNA nanoballs.

[0041] In the fifth aspect of the present invention, the present invention provides a cell dispersion, comprising: cells and a second carrier; the second carrier carries a plurality of droplet identification molecules, the plurality of droplet identification molecules can be connected to the second carrier via covalent bonds or other means, and the droplet identification molecules carry a droplet label sequence.

[0042] According to an embodiment of the present invention, the cell dispersion provided in the fifth aspect may further include the following technical features:

[0043] According to an embodiment of the present invention, the cell is in a single cell form.

[0044] According to an embodiment of the present invention, the droplet identification molecule further includes a recognition sequence of a capture sequence, and the recognition sequence of the capture sequence is connected to the droplet tag sequence.

[0045] According to an embodiment of the present invention, the covalent bond is a disulfide bond.

[0046] In the sixth aspect of the present invention, the present invention provides a sequencing method, comprising: sequencing the sequencing library described in any embodiment of the first aspect of the present invention to obtain a sequencing result consisting of a plurality of sequencing reads; and classifying sources of the plurality of sequencing reads based on at least one of the cell label sequence, the droplet label sequence and the molecular label sequence.

[0047] In the seventh aspect of the present invention, the present invention provides a circular nucleic acid molecule, comprising: a replication initiation sequence; a capture sequence recognition sequence; and a droplet label sequence. The circular nucleic acid molecule provided by the present invention includes a replication initiation sequence that can be used for replication and amplification, and also contains a capture sequence recognition sequence for being recognized by a capture sequence from a carrier; and also contains a droplet label sequence that can be applied to a droplet as a marker indicating sequencing reads from the same droplet.

[0048] According to an embodiment of the present invention, the circular nucleic acid molecule further comprises: an endonuclease recognition sequence. The endonuclease recognition sequence can be recognized by an endonuclease, thereby facilitating subsequent acquisition of a large number of identical chain nucleic acid molecules.

[0049] In the eighth aspect of the present invention, the present invention provides a method for preparing a long-chain molecule, comprising: performing rolling circle replication amplification on the circular nucleic acid molecule of the sixth aspect of the present invention to obtain the long-chain molecule, thereby obtaining the long-chain molecule conveniently and quickly.

[0050] In the ninth aspect of the present invention, the present invention provides a linear nucleic acid molecule, comprising: a 5' terminal sequence and a 3' terminal sequence; a replication initiation sequence; a capture sequence recognition sequence; and a droplet tag sequence; wherein the 5' terminal sequence and the 3' terminal sequence are suitable for jointly forming an endonuclease recognition sequence.

[0051] In the tenth aspect of the present invention, the present invention provides a method for analyzing single-cell nucleic acid, comprising: providing a single-cell suspension, the single-cell suspension containing single dispersed cells, mixing the single-cell suspension with a droplet identification molecule to obtain a cell suspension, the droplet identification molecule carrying a droplet label sequence; placing the cell suspension, a first carrier and oil at different positions of a microfluidic chip, respectively, so that the cell suspension, the first carrier and the oil pass through the same channel to obtain droplets, the droplets being the droplets described in any embodiment of the second aspect of the present invention; performing demulsification treatment and library construction on the droplets to obtain a sequencing library; and performing sequencing analysis on the sequencing library to obtain the nucleic acid information of the single cell.

[0052] According to an embodiment of the present invention, the above method for analyzing single cell nucleic acid may further include the following technical features:

[0053] According to an embodiment of the present invention, the concentration of the single cell suspension is 100 to 200 cells per microliter, and the concentration of the droplet identification molecule is 10 5 ~10 8 The copy number of the droplet identification molecule.

[0054] According to an embodiment of the present invention, the concentration of the carrier is 2000 to 3000 carriers per microliter.

[0055] The technical solution provided by the present invention can reduce the input of cell samples for droplet microfluidics single-cell library construction to 10,000, and the cell recovery rate can reach more than 50%. The number of usable cells obtained in a single experiment is 4,000-6,000, which greatly reduces the cost of single-cell library construction and sequencing, and is conducive to the development of large-scale single-cell research projects.

[0056] Additional aspects and advantages of the present invention will be given in part in the following description and in part will be obvious from the following description, or will be learned through practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0057] The above and / or additional aspects and advantages of the present invention will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

[0058] Figure 1 is a schematic diagram of a droplet provided according to an embodiment of the present invention;

[0059] Figure 2 is a schematic diagram of a droplet provided according to an embodiment of the present invention;

[0060] Figure 3is a schematic diagram of a DNA oligo sequence provided according to an embodiment of the present invention;

[0061] Figure 4 is a schematic diagram of a microfluidic chip provided according to an embodiment of the present invention;

[0062] Figure 5 is a diagram of the quality inspection results of the cDNA product provided in Example 1 of the present invention;

[0063] Figure 6 This is a quality inspection result diagram of a DNB oligo amplification product containing a droplet tag sequence provided in Example 1 of the present invention;

[0064] Figure 7 is a graph showing the quality inspection results of the transcriptome library provided in Example 1 of the present invention;

[0065] Figure 8 This is a diagram showing the quality inspection results of a DNB oligo library containing a droplet tag sequence provided in Example 1 of the present invention;

[0066] Fig. 9 This is a result graph of detecting the number of cells and the number of genes in a single cell according to the sequencing results of the transcriptome library provided in Example 1 of the present invention;

[0067] Fig.10 is the number of cell labels corresponding to each DNB oligo provided in Example 1 of the present invention;

[0068] Fig.11 is a correlation analysis result diagram provided according to Example 1 of the present invention;

[0069] Fig.12 is a diagram of the cell barcode pairing analysis results provided in Example 1 of the present invention;

[0070] Fig.13 is a diagram of the quality inspection results of the cDNA product provided in Example 2 of the present invention;

[0071] Fig.14 This is a quality inspection result diagram of a DNB oligo library containing a droplet tag sequence provided in Example 2 of the present invention;

[0072] Fig.15 This is a result graph of detecting the number of cells and the number of genes in a single cell according to the sequencing results of the transcriptome library provided in Example 2 of the present invention;

[0073] Fig.16 The molecular quantity result of the captured droplet label sequence corresponding to each combination of cell label sequence and droplet label sequence provided in Example 2 of the present invention;

[0074] Fig.17 is the number of types of droplet label sequences corresponding to each cell label sequence provided according to Example 2 of the present invention.

[0075] Fig.18 is the number of cell label sequences corresponding to each droplet label sequence provided in Example 2 of the present invention;

[0076] Fig.19 is a correlation analysis result diagram provided according to Example 2 of the present invention;

[0077] Fig. 20 This is a diagram of the cell barcode pairing analysis results provided according to Example 2 of the present invention. DETAILED DESCRIPTION

[0078] Embodiments of the present invention are described in detail below, and examples of the embodiments are shown in the accompanying drawings. The embodiments described below with reference to the accompanying drawings are exemplary and intended to be used to explain the present invention, but should not be understood as limiting the present invention.

[0079] In the process of describing the present invention, the relevant terms in the text are explained and illustrated. These explanations and illustrations are only for the convenience of understanding the scheme and cannot be regarded as limitations on the protection scheme of the present invention.

[0080] Herein, when referring to nucleic acid molecules, it may refer to RNA molecules or DNA molecules, and may be modified DNA molecules and / or RNA molecules or unmodified DNA molecules and / or RNA molecules. A person skilled in the art may specifically judge whether it is an RNA molecule or a DNA molecule, or both, based on the context and context. In addition, when referring to nucleic acid molecules being connected, unless otherwise specified, one of the most common ways of connecting is that they can be connected via a 3'-5' phosphodiester bond. Of course, unless otherwise specified, when these different nucleic acid molecules are connected, they are not required to be directly connected, and may also be connected via other nucleic acid molecules, as long as they are on the same nucleic acid chain or on the same ring.

[0081] In this article, whether it is a "cell tag sequence", "droplet tag sequence" or "capture sequence identification sequence", etc., it is a nucleic acid molecule. As mentioned above, these nucleic acid molecules can be RNA molecules or DNA molecules. Different expressions are only used to represent the different functions of these nucleic acid sequences. Those skilled in the art can understand the role of the sequences represented by various expressions according to the context.

[0082] For example, in this article, a cell barcode can be used to indicate that the sequencing reads come from the same cell. A drop barcode can indicate that the sequencing reads come from the same droplet. A capture sequence refers to a sequence that can be used to match other nucleic acid molecules and to capture other nucleic acid molecules. A capture sequence recognition sequence refers to a sequence that can be recognized by a capture sequence. Usually, this sequence can be matched with a capture sequence to achieve the capture of nucleic acid molecules.

[0083] In order to solve the defect that a large amount of cell samples are needed when performing single-cell library sequencing based on a droplet microfluidic platform, the present invention allows multiple microbeads to be encapsulated in one droplet when preparing droplets, and then the sequencing reads from the same droplet in these microbeads are judged to improve the utilization rate of cell samples.

[0084] According to an embodiment of the present invention, the provided droplets may contain biological materials, droplet identification molecules and a first carrier at the same time, the biological materials contain nucleic acid molecules, the droplet identification molecules carry a droplet label sequence, the first carrier carries a capture sequence and a cell label sequence, the capture sequence is used to capture at least one of the nucleic acid molecules and the droplet identification molecules. The first carrier may further carry an amplification recognition sequence.

[0085] According to an embodiment of the present invention, the droplet identification molecule in the droplet can be obtained by means of the DNB technique, such as Figure 1 For example, a DNA fragment carrying a droplet tag sequence, an endonuclease recognition sequence, a PCR adapter sequence and poly A can be synthesized, and the fragment length is between 100-2000 bp; then, an oligonucleotide that can be anchored on the DNA fragment can be used as a primer to perform rolling circle linear amplification, and finally a DNA fragment (DNA nanoball) containing 20-200 copies can be obtained.

[0086] Of course, since the droplet identification molecules obtained by means of the DNB technology are provided in the form of single strands, and since the endonuclease recognizes double-stranded nucleic acid molecules, the oligonucleotides that can be hybridized with the above endonuclease recognition sequence can be hybridized with the endonuclease recognition sequence; this facilitates the subsequent enzymatic treatment with the endonuclease to obtain a large number of the same droplet identification molecules.

[0087] In addition, the DNA nanospheres and biomaterials can be pre-mixed, the vectors and endonucleases can be pre-mixed, and then the droplets can be encapsulated using a conventional droplet microfluidics platform. The DNA nanospheres are broken into small fragments by the restriction endonucleases and captured by the vectors (such as microbeads) with poly T. The vectors from the same droplet will capture the same droplet tag sequence. Figure 1As shown, each droplet formed contains different cells and DNA with different droplet label sequences. After the subsequent reaction is completed, based on the similarity of the droplet label sequences on the beads, it is possible to distinguish which beads come from the same droplet, that is, the nucleic acid information corresponding to the same cell.

[0088] According to an embodiment of the present invention, the concentration of DNA nanospheres is 10 5 ~10 8 The number of copies is set to a certain value, so that a single droplet contains at least one DNA nanoball. Under this concentration condition, a single droplet can contain more than one DNA nanoball; there are also cases where a small number of droplets contain more than two DNA nanoballs. In this case, multiple magnetic beads in the same droplet can capture two droplet label sequences, and multiple magnetic beads in other droplets can capture other droplet label sequences. Combined with the correlation of multiple nanoballs being captured by multiple magnetic beads in the droplet at the same time, it is possible to calculate which magnetic beads are from the same droplet.

[0089] According to an embodiment of the present invention, the droplet identification molecule in the droplet can also be provided in the form of a carrier. In order to distinguish it from the first carrier that provides the cell label sequence, it is referred to as the second carrier here, that is, the droplet identification molecule can be provided in the form of a second carrier, and the second carrier carries a plurality of droplet identification molecules, and the plurality of droplet identification molecules can be connected to the second carrier by covalent bonds or other means. The droplet identification molecule carries the droplet label sequence, and can further contain the recognition sequence of the capture sequence, and the recognition sequence of the capture sequence is connected to the droplet label sequence. The droplet identification molecule is connected to the carrier by a covalent bond, for example, it can be connected to the carrier by a disulfide bond, which is convenient for the subsequent use of some covalent bond breaking reagents, such as DTT to destroy the covalent bond, release the droplet identification molecule, and thus facilitate the capture of the capture sequence carried on the first carrier. Or the second carrier can be a hydrogel microparticle, and the plurality of droplet identification molecules can be embedded in the hydrogel microparticles, and the hydrogel is melted under suitable reaction conditions to release the droplet identification molecule.

[0090] According to an embodiment of the present invention, generally speaking, the available second carrier can be a magnetic bead, and the size of the magnetic bead can generally be smaller than the size of the magnetic bead of the first carrier. The second carrier carries multiple identical droplet label sequences. When droplets are subsequently formed, the covalent bond breaking reagent in the droplet can destroy the covalent connection between the droplet label sequence and the second carrier without affecting the hydrogen bond connection between nucleic acid molecules, thereby releasing a large number of droplet label sequences in the droplet. The large number of released droplet label sequences will be captured by the first carrier, and the carriers from the same droplet will capture the same droplet label sequence. Figure 2As shown, each droplet formed contains different cells and DNA with different droplet label sequences. After the subsequent reaction is completed, based on the similarity of the droplet label sequences on the beads, it is possible to distinguish which beads come from the same droplet, that is, the nucleic acid information corresponding to the same cell.

[0091] Conventional droplet microfluidics technology is then used to perform emulsion breaking, reverse transcription reaction, enzyme digestion of empty oligonucleotides on the surface of the carrier, cDNA amplification, library construction, sequencing and analysis, thereby completing single-cell sequencing.

[0092] The scheme of the present invention will be explained below in conjunction with the embodiments. It will be appreciated by those skilled in the art that the following embodiments are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. Where specific techniques or conditions are not indicated in the embodiments, the techniques or conditions described in the literature in this area or the product specifications are used. The reagents or instruments used are not indicated by the manufacturer and are all conventional products that can be obtained commercially.

[0093] Example 1

[0094] Example 1 provides a method for preparing a sequencing library and performing high-throughput sequencing. The provided method is obtained through several steps, including: DNA nanoball preparation, single cell suspension preparation, microbead preparation, droplet generation, emulsion breaking, reverse transcription RT reaction, enzyme digestion, cDNA amplification, fragmentation enzyme library construction, high-throughput sequencing, etc.

[0095] 1. Preparation of DNA nanospheres

[0096] Reference Figure 3 As shown, firstly, the DNB oligo sequence is synthesized, and then cyclized to prepare DNB. Then, by combining with the primer complementary to the restriction site, the endonuclease recognition sequence on the DNB is annealed into a double-stranded sequence, which is convenient for subsequent digestion with the endonuclease.

[0097] 1.1 Primer sequence synthesis

[0098] The following sequence was artificially synthesized:

[0099] DNB oligo sequence:

[0100]

[0101] Note: The UMI restriction site is split at both ends of the sequence (they will be connected after cyclization) + a number of restriction protection bases are added at both ends of the restriction site + Poly T sequence (Poly A sequence is formed after making DNB) + fixed sequence + DNB UMI + PCR adapter sequence (because the library cannot be interrupted, this adapter can be recognized and amplified during Sample index amplification).

[0102] Specifically, the single underlined bold in the above sequence and" GGTCT " is the restriction endonuclease cleavage site of BsaI, which is split at both ends of the sequence (will be connected after cyclization). The dotted line in the above sequence and It is the enzyme protection base to improve the efficiency of enzyme cutting. The "TTTTTTTTTTTTTTTTTTTTTTTV" in the above sequence is synthesized into a Poly A sequence after DNB, which can be complementary to the oligonucleotide sequence on the magnetic bead for capture. The 10nt base "N" in the sequence is a random sequence for marking droplets (that is, the droplet label sequence mentioned in the article), so that each DNB carries a different label sequence. It is a PCR adapter, which can be complementary to the library adapter PCR primer for amplification during library construction.

[0103] Splint oligo:

[0104] 5'-GTATTATTAGAGACCTATCT-3'

[0105] Among them, Splint oligo is a circular complementary primer.

[0106] Digestion site complementary oligo:

[0107]

[0108] Description: The 5' and 3' ends of the synthesized DNB-oligo sequence contain restriction site sequences, which need to be circularized. After the circularization, the complete restriction site is pieced together to form a DNB-oligo ring. The formed DNB-oligo ring is then used for rolling circle replication and amplification to form a DNA nanoball (DNB). The generated single strand is the complementary strand of the DNB-oligo ring, and then the restriction site complementary sequence is added, so the complementary sequence is consistent with the two end sequences of the original oligo.

[0109] 1.2 Single-chain DNA oligocyclization

[0110] a. Take 200-400 ng of DNB oligo sequence into a new 0.2 ml PCR tube and add TE Buffer (Cat. No.: AM9849) to a total volume of 47 μl.

[0111] b. Add 3.0 μl 20 μM Splint oligo, mix well and centrifuge briefly to the bottom of the tube.

[0112] c. Place in PCR instrument and incubate at 95°C for 3 min.

[0113] d. After the reaction is completed, immediately transfer the PCR tube to ice and let it stand for 5 minutes.

[0114] e. Prepare single-stranded circularization reaction solution on ice: 3.2μl TE Buffer, 6μl 10x TA buffer (Cat. No.: Y038), 0.6μl 100mM ATP (Cat. No.: R1441), 0.2μl 600U / μl T4 DNA Ligase (Cat. No.: BGE004).

[0115] f. Use a pipette to draw 10 μl of the prepared single-stranded circularization reaction solution and add it to the above PCR tube. Vortex and shake to mix well. Centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0116] g. Place the PCR tube on a PCR instrument, incubate at 37°C for 30 min, and heat the cover at 75°C.

[0117] h. Prepare enzyme digestion reaction solution on ice: 1.0μl TE Buffer, 0.4μl 10x TA buffer, 1.95μl 20U / μl EXO I (Cat. No.: 01E010MS), 0.65μl 100U / μl EXO III (Cat. No.: 01E011HS).

[0118] i. Pipette 4 μl of the enzyme digestion reaction solution into the single-stranded cyclization product, vortex to mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0119] j. Place in a PCR instrument, incubate at 37°C for 30 min, and heat cover at 75°C. Use the enzyme digestion reaction solution to digest and degrade the single-stranded oligonucleotide chain that has not been circularized.

[0120] k. After the reaction is completed, add 3 μl 0.5 M EDTA (Cat. No.: AM9260), mix well and centrifuge.

[0121] l. Use 90 μl PEG32 (Cat. No.: Y041) magnetic beads to recover the above enzyme digestion products, use 32 μl TEBuffer to elute the DNA, and use ssDNA Assay Kit (Cat. No.: Q10212) was used to detect the concentration of single-stranded products.

[0122] 1.3 RCA and restriction site hybridization reaction

[0123] a. Use the make DNB kit (Cat. No.: 1000012552) to prepare the following reaction system on ice: 4 μl single-stranded product ssDNA (10 ng), 4 μl 10X Phi29 Buffer, 1.0 μl 20 μM Splint oligo, 16 μl TE Buffer, 15 μl H2O.

[0124] b. Mix the above 40 μl reaction mix by vortexing and centrifuging in a mini centrifuge for 5 seconds.

[0125] c. Place on PCR instrument, 95℃ for 1 min, 65℃ for 1 min, 40℃ for 1 min, heat cover at 105℃.

[0126] d. Take out the DNB polymerase mixture II (LC), centrifuge briefly for 5 seconds, and place it in an ice box for later use.

[0127] e. After the reaction in step c is completed, take out the PCR tube and add 40μl make DNB enzyme Mix and 4μl make DNB enzyme Mix II on ice.

[0128] f. Use a vortex shaker to mix well and centrifuge in a mini centrifuge for 5 seconds.

[0129] g. Place the tube on a PCR instrument and incubate at 30°C for 20 min. When the temperature drops to 4°C, immediately stop the task and transfer the PCR tube to an ice box. Add 20 μl of DNB termination buffer and slowly mix with a wide-mouth pipette tip for 5-8 times. Do not shake or centrifuge.

[0130] h. Use ssDNA Assay Kit was used to detect the concentration of DNB.

[0131] i. Take 20μl DNB of the above reaction product to a new PCR tube, add 10μl 10uM Digestion sitecomplementary oligo, 10μl 10x Cutsmart buffer (Cat. No.: B7204S), 60μl H2O, and gently pipette 5-8 times. Do not shake and centrifuge.

[0132] j. Place on the PCR machine, 55℃ for 2min, 40℃ for 2min, 30℃ for 2min, 20℃ for 2min, 10℃ for 2min, 4℃ for 2min.

[0133] Note: This step is the annealing of complementary primers at the restriction site. Generally, when sequencing on a sequencer, the complementary primers on the DNB are at 55 degrees, so a 55-degree gradient annealing strategy is used.

[0134] k. Take 10 μl of the above product into a new PCR tube, add 90 μl EB buffer to dilute, quantify, and place on ice for later use.

[0135] 2. Single Cell Suspension Preparation

[0136] 2.1 For 293T cell lines and NIH3T3 cell lines, single cell suspension was prepared by trypsin digestion, and washed 1-2 times with PBS (Cat. No.: 10010031) (containing 0.04% BSA), and filtered using a 40 μm cell sieve.

[0137] 2.2 Use a cell counting plate or a cell counter to detect the cell concentration.

[0138] 2.3 Depending on the cell concentration, aspirate 20,000 cells each time, collect the cell pellet by centrifugation at 300-500g, and add 100μl Cell Resuspension Buffer to resuspend the cells.

[0139] 2.4 Before loading, add 10 μl of the DNB product obtained in step k of step 1.3 above to the Cell Resuspension Buffer, mix evenly with a widened pipette tip, and place on ice until loading.

[0140] 3. Microbead preparation

[0141] 3.1 Refer to the article published by BGI on the preparation method of stLFR magnetic beads (Efficient and unique co-barcoding of second-generation sequencing reads from long DNA molecules enabling cost effective and accurate sequencing, haplotyping, and de novoassembly, https: / / genome.cshlp.org / content / early / 2019 / 04 / 02 / gr.245126.118 ) Magnetic beads (purchased from Spherotech, USA, item number SVM-200-4, https: / / www.spherotech.com / coa_mag_ par.htm ) to embed the surface oligonucleotides to obtain magnetic beads with oligonucleotides on the surface. Pipette 200 μl (220,000) of the magnetic beads into a 0.2 ml PCR tube, place it on a magnetic stand and let it stand for 2 minutes, and remove the supernatant.

[0142] 3.2 Remove the PCR tube from the magnetic stand and add 200 μl 1x Buffer D (1 mM EDTA, 9 mg / ml 85% KOH) to suspend the magnetic beads.

[0143] 3.3 Incubate at room temperature for 5 minutes.

[0144] 3.4 Place on the magnetic rack and let stand for 2 minutes, then remove the supernatant.

[0145] 3.5 Keep the PCR tube on the magnetic stand, add 200 μl 1x Buffer D, let stand for 30 seconds and remove the supernatant.

[0146] 3.6 Add 200 μl LSWB, let stand for 30 seconds, and then remove the supernatant.

[0147] 3.7 Repeat the previous step.

[0148] 3.8 Add 200μl Lysis Buffer (6% Ficoll PM-400 (Cat. No.: 17-0300-10), 0.2% Sarkosyl (Cat. No.: L7414), 20mM EDTA, 200mM Tris pH 7.5 (Cat. No.: T2944-1L), H2O), let stand for 30s, and then remove the supernatant. Remove the PCR tube from the magnetic stand, add 100μl Lysis Buffer and 5.5μl 1M DTT, and finally add 4.5μl BsaI (Cat. No.: R0535S) restriction enzyme, suspend the magnetic beads with a low-adsorption pipette tip and place on ice.

[0149] 4. Droplet generation

[0150] 4.1 Remove the protective film on the chip surface and place it in the chip slot area of ​​the droplet generator. Figure 4 shown.

[0151] 4.2 Insert the A end of the connecting tube on the collection cover (the connecting tube that touches the bottom of the collection tube) into the Outlet hole of the chip.

[0152] 4.3 Place the 50ml syringe on the fixed stand and adjust the push rod to the initial position. Use a flat needle to connect the syringe to the connecting tube B end on the collection tube cover (the connecting tube that does not touch the bottom of the collection tube).

[0153] 4.4 Add 200 μl of droplet generation oil to the collection tube, tighten the collection cap, and place the collection tube vertically on the fixed stand.

[0154] 4.5 Use a pipette to gently blow and mix the cells, and add 100 μl of the single-cell suspension (obtained in step 2 above) to the cells well of the chip, making sure the pipette tip touches the bottom of the well.

[0155] 4.6 Use a pipette to gently blow and mix the magnetic beads, and add 100 μl of magnetic beads (obtained in step 3 above) to the beads well of the chip, making sure the pipette tip touches the bottom of the well.

[0156] 4.7 Immediately add 350 μl of droplet generation oil to the Oil well of the chip.

[0157] 4.8 Quickly pull the push rod of the syringe to the slot position and lock the push rod in the slot.

[0158] 4.9 Start the timer for 8 minutes and collect the droplets.

[0159] 4.10 After 8 minutes, immediately loosen the collection cap on the collection tube, pull out the connecting tube from the chip outlet hole, stretch the connecting tube vertically to allow the droplets in the tube to flow into the collection tube, and then replace the ordinary collection tube cap.

[0160] 4.11 Let stand at room temperature for 20 minutes to allow the mRNA molecules to fully bind to the magnetic beads.

[0161] 5. Demulsification

[0162] 5.1 Prepare the demulsification reagent by adding 10 ml 6X SSC (Cat. No.: 15557-036) and 200 μl PFO (Cat. No.: 370533-25G) into a 15 ml centrifuge tube.

[0163] 5.2 Connect the filter device to the vacuum pump, adjust the pressure parameter to 0.01MPa or 100mbar, and start the vacuum pump.

[0164] 5.3 Add 20 ml of 6X SSC to pre-treat the device.

[0165] 5.4 When there is no liquid left on the filter membrane, pour all the liquid in the collection tube evenly onto the surface of the filter membrane, and wash the collection tube twice with 2ml 6X SSC, and pour the washing solution into the filtration device.

[0166] 5.5 Mix 10 ml of demulsification reagent by inverting vigorously and quickly pour it into the filter device.

[0167] 5.6 When there is no liquid residue on the filter membrane, add 30 ml of 6X SSC to wash the magnetic beads.

[0168] 5.7 When there is no liquid residue on the filter membrane, turn off the vacuum pump and disconnect the vacuum pump from the filtration device.

[0169] 5.8 Seal the filter port of the filter device with a syringe or rubber stopper.

[0170] 5.9 Use a pipette to add 1.0 ml of collection buffer and gently blow over the entire filter surface about 20 times to suspend the magnetic beads.

[0171] 5.10 Transfer the collected solution containing magnetic beads to a 1.5 ml low-binding centrifuge tube.

[0172] 5.11 Use 1.0 ml of collection buffer to gently blow over the entire filter membrane surface about 10 times to suspend the remaining magnetic beads.

[0173] 5.12 Transfer the collected solution containing magnetic beads to a 1.5 ml low-adsorption centrifuge tube, place it on a magnetic rack, let it stand for 2 minutes, and slowly remove the supernatant.

[0174] 5.13 Remove the centrifuge tubes from the magnetic stand, use 100 μl of collection buffer to suspend the magnetic beads adsorbed on one side of the two centrifuge tubes in turn, and transfer the liquid to a 0.2 ml low-adsorption PCR tube.

[0175] 5.14 Use 100 μl of collection buffer again to suspend the magnetic beads adsorbed on one side of the two centrifuge tubes in turn, and transfer the liquid to the above 0.2 ml low-adsorption PCR tube.

[0176] 5.15 Place the PCR tube containing magnetic beads on the magnetic rack, let it stand for 2 minutes, and remove the supernatant.

[0177] 5.16 Keep the magnetic beads adsorbed, add 200 μl 6X SSC, let stand for 30 seconds, and remove the supernatant.

[0178] 5.17 Add 200 μl 5X FS Buffer, let stand for 30 seconds, and slowly remove the supernatant to avoid aspirating the magnetic beads.

[0179] 6. Reverse transcription reaction

[0180] 6.1 Prepare the reverse transcription reaction system on ice: 5μl H2O, 20μl 5x First-Strand Buffer (Cat. No.: 01E022MS), 20μl 5M Betaine (Cat. No.: B0300-1VL), 10μl 10mM dNTPs (Cat. No.: N0447L), 7.5μl 100mM MgCl2 (Cat. No.: 20-303), 5μl 50μM Template switch oligo, 5μl 100mM DTT (Cat. No.: 01E022MS), 5μl 200U / μl Alpha reverse transcriptase (Cat. No.: 01E022MS), 2.5μl 40U / μl RNase inhibitor (Cat. No.: 01E019MS).

[0181] The Alpha reverse transcriptase is an engineered MMLV reverse transcriptase, which can recognize ssDNA as a template for complementary synthesis.

[0182] 6.2 Pipette 100 μl of reverse transcription reaction system, add it to the PCR tube containing magnetic beads, and mix by pipetting.

[0183] 6.3 Perform reverse transcription reaction under the following conditions: 42°C, 90 min; 10 cycles (50°C, 2 min; 42°C, 2 min), with the hot cover set to 75°C. Due to the sedimentation of magnetic beads, flick the beads to mix them every 20 min, centrifuge briefly and continue the reaction.

[0184] 6.4 After the reaction is completed, centrifuge briefly, place on a magnetic rack, let stand for 2 minutes, and remove the reaction solution.

[0185] 6.5 Remove the PCR tube from the magnetic rack, add 200 μl TE-SDS, shake and mix, and terminate the reaction.

[0186] 6.6 After a brief centrifugation, place on a magnetic rack, let stand for 2 minutes, and remove the liquid.

[0187] 6.7 Keep the magnetic beads adsorbed, add 200 μl TE-TW, let stand for 30 seconds, and remove the supernatant.

[0188] 6.8 Repeat the above steps.

[0189] 6.9 Keep the magnetic beads adsorbed, add 200 μl 10 mM Tris pH 8.0, let stand for 30 seconds and remove the supernatant.

[0190] 7. Enzyme digestion of empty oligos that do not capture mRNA molecules on the surface of microbeads

[0191] 7.1 Enzyme digestion reaction system: 170μl H2O, 20μl 10x EXO I Buffer, 10μl EXO I enzyme.

[0192] 7.2 Pipette 200 μl of enzyme digestion reaction system, add it to the PCR tube containing magnetic beads, and vortex to mix.

[0193] 7.3 After a brief centrifugation, place in a PCR instrument and incubate at 37°C for 45 minutes with the heated lid set to 75°C. Gently flick the beads every 15 minutes to mix them well, briefly centrifuge and continue the reaction.

[0194] 7.4 After the reaction is completed, centrifuge briefly, place on a magnetic rack, let stand for 2 minutes, and remove the reaction solution.

[0195] 7.5 Remove the PCR tube from the magnetic stand, add 200 μl TE-SDS, shake and mix, and terminate the reaction.

[0196] 7.6 After a brief centrifugation, place on a magnetic rack, let stand for 2 minutes, and remove the liquid.

[0197] 7.7 Keep the magnetic beads adsorbed, add 200 μl TE-TW, let stand for 30 seconds, and remove the supernatant.

[0198] 7.8 Remove the PCR tube from the magnetic stand and add 200 μl TE-TW to suspend the magnetic beads.

[0199] 7.9 After a brief centrifugation, place on a magnetic rack, let stand for 2 minutes, and remove the liquid.

[0200] 7.10 Keep the magnetic beads in adsorption state, add 200 μl H2O, let stand for 30 seconds, and remove the supernatant.

[0201] 8. cDNA Amplification

[0202] 8.1 Prepare PCR reaction system: 41 μl H2O, 8 μl 10 μM Tn Primer, 2 μl 1 μM reverse primer, 50 μl 2x KAPA HiFi Hotstart Ready mix (Cat. No. KK2602).

[0203] 8.2 Carry out PCR reaction under the following conditions: 95°C, 3 min; 13-20 cycles (98°C, 20 s; 58°C, 20 s; 72°C, 3 min).

[0204] 8.3 After PCR, use 60 μl (0.6X) AMPure XP Beads (Cat. No.: A63881) (equilibrated at room temperature for 30 min in advance) to purify and recover the PCR product. Use 200 μl (2X) AMPure XP Beads to recover the oligo fragment product in the supernatant and use Qubit dsDNA HS Kit (Cat. No.: Q32854) to detect the concentration.

[0205] 8.4 Prepare the PCR reaction system for secondary amplification of the DNB-Oligo purified product: 11μl H2O, 2μl 10μM V4-Phos-Tn-C Primer, 2μl 10μM V2-N7-index-n Primer, 25μl 2x KAPA HiFi HotstartReady mix, 10μl DNB-Oligo purified product in step 8.3.

[0206] 8.5 Carry out PCR reaction under the following conditions: 95°C, 3 min; 6-10 cycles (98°C, 20 s; 58°C, 20 s; 72°C, 15 s).

[0207] 8.6 Use 200 μl (2X) AMPure XP Beads to recover the oligo secondary amplification product and detect the concentration using Qubit dsDNA HSKit.

[0208] 9. Fragmentation enzyme library construction

[0209] 9.1 Take out the fragmentase, shake and mix for 5 seconds, centrifuge briefly and place on ice for later use.

[0210] 9.2 Take 50-300ng of the cDNA to be sheared and put it into a new 0.2ml PCR tube. The volume should be ≤16μl. The part less than 16μl should be supplemented with H2O.

[0211] 9.3 Use a pipette to draw 4.0 μl of the prepared fragmentation reaction solution (2 μl fragmentase (Cat. No.: M0348L), 2 μl 10x fragmentase buffer (Cat. No.: B0349)) into the cDNA tube, vortex for 3 times, 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0212] 9.4 Place the PCR tube on the PCR instrument and incubate at 37℃ for 10 minutes.

[0213] 9.5 Add 30 μl 0.1 M EDTA to the PCR tube, vortex to mix, and terminate the reaction.

[0214] 9.6 Centrifuge briefly to collect the reaction solution at the bottom of the tube and place it on ice.

[0215] 9.7 Use 0.6x+0.4x (i.e. 30μl+20μl) AMPure XP Beads to select the fragments of the fragmented products and use 42μl H2O to elute the DNA.

[0216] 9.8 Prepare end repair reaction solution on ice: 2.3μl H2O, 5μl 10x PNK buffer (Cat. No.: B9040L), 1.2μl 5:1 dATP:dNTP mix, 0.6μl 10U / μl T4 Polynucleotide Kinase (Cat. No.: Y9040L), 0.6μl 3U / μl T4 DNA polymerase (Cat. No.: P7080L), 0.2μl 5U / μl rTaq (Cat. No.: R500Z), 0.1μl 5U / μl Klenow fragment (Cat. No.: P7060L).

[0217] 9.9 Use a pipette to draw 10 μl of the end repair reaction solution and add it to the screened fragmentation product. Vortex to mix well and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0218] 9.10 Place the PCR tube in a PCR instrument and incubate at 37°C for 30 min or 65°C for 15 min.

[0219] 9.11 After the reaction is completed, place the reaction solution on ice.

[0220] 9.12 Prepare adapter ligation reaction solution on ice: 3.6μl H2O, 3μl 10x PNK buffer, 5μl 10μM Adapters mix, 0.8μl 100mM ATP (Cat. No.: R1441), 16μl 50% PEG 8000 (Cat. No.: EB-0.5P8K-250), 1.6μl 600U / μl T4 DNA Ligase (Cat. No.: BGE004).

[0221] 9.13 Use a pipette to slowly draw up 30 μl of the prepared adapter ligation reaction solution, add it to the end repair product, vortex to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0222] 9.14 Place the PCR tube on the PCR instrument and incubate at 23℃ for 60 minutes.

[0223] 9.15 After the reaction is completed, add 20 μl TE Buffer to make the total sample volume reach 100 μl.

[0224] 9.16 Use 50 μl AMPure XP Beads to purify and recover the ligation product, and use 48 μl H2O to elute the ligation product.

[0225] 9.17 Add 2 μl 10 μM PCR Primer and 50 μl 2x KAPA HiFi Hotstart Ready mix and vortex to mix.

[0226] 9.18 Carry out PCR reaction according to the following conditions: 95°C, 3 min; 11 cycles (98°C, 20 s; 58°C, 20 s; 72°C, 30 s).

[0227] 9.19 After PCR, use 0.6x+0.2x (i.e. 60μl+20μl) AMPure XP Beads to screen the PCR products, elute the DNA with 42μl TE Buffer, and detect the concentration using Qubit dsDNA HS Kit.

[0228] 10. High-throughput sequencing

[0229] 10.1 Take a total of 200-400ng of library into a new 0.2ml PCR tube, where the pooling ratio of the cDNA sheared PCR product and the oligo secondary PCR product of the same sample is 9:1 (you can also circularize them separately and then pool them). Use TEBuffer to add to a total volume of 47μl.

[0230] 10.2 Add 3.0 μl 20 μM Splint Oligo primer, mix well and centrifuge briefly to the bottom of the tube.

[0231] 10.3 Place in PCR instrument and incubate at 95℃ for 3 minutes.

[0232] 10.4 After the reaction is completed, immediately transfer the PCR tube to ice and let it stand for 5 minutes.

[0233] 10.5 Prepare single-strand circularization reaction solution on ice: 3.2μl TE Buffer, 6μl 10x TA buffer, 0.6μl 100mM ATP, 0.2μl 600U / μl T4 DNA Ligase.

[0234] 10.6 Use a pipette to draw 10 μl of the prepared single-stranded circularization reaction solution and add it to the above PCR tube. Vortex to mix well and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0235] 10.7 Place the PCR tube on the PCR instrument and incubate at 37℃ for 30 minutes.

[0236] 10.8 Prepare the enzyme digestion reaction solution on ice: 1.0μl TE Buffer, 0.4μl 10x TA buffer, 1.95μl 20U / μl EXO I, 0.65μl 100U / μl EXO III.

[0237] 10.9 Pipette 4 μl of the enzyme digestion reaction solution and add it to the single-stranded cyclization product, vortex to mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0238] 10.10 Place the tube on a PCR instrument and incubate at 37°C for 30 minutes.

[0239] 10.11 After the reaction is completed, add 3 μl 0.5 M EDTA, mix well and centrifuge.

[0240] 10.12 Use 90 μl PEG32 magnetic beads to recover the above enzyme digestion products, use 32 μl TE Buffer to elute the DNA, and use ssDNA Assay Kit detects the concentration of single-stranded circular library.

[0241] 10.13 The qualified libraries were sequenced using a sequencing machine with the sequencing parameters being 41+100+10.

[0242] The quality inspection results of the experimental products and libraries are as follows:

[0243] Figure 5 The quality inspection result of the obtained cDNA product is shown in FIG. Figure 6 The figure shows the quality inspection results of the DNBoligo amplification product containing the droplet tag sequence. Figure 7 The figure shows the quality inspection results of the obtained transcriptome library. Figure 8 The figure shows the quality inspection results of the obtained DNB oligo library containing the droplet tag sequence.

[0244] After sequencing analysis, the results are shown in Table 1 and Fig. 9 As shown:

[0245] Table 1 Transcriptome sequencing results

[0246] Estimated number of cell 1689 Reads in cell 0.421 Mean UMI counts per cell 11710 Mean genes per cell 3441

[0247] Table 1 and Fig. 9 The results of analyzing the number of cells and the number of genes in a single cell based on the results of transcriptome sequencing are shown. Fig. 9 Here UB represents the number of UMI counts, and GN represents the number of genes.

[0248] Fig.10 The number of cell labels corresponding to each DNB oligo is shown. From this figure, it can be seen that the number of barcodes corresponding to each DNB-oligo is mostly 1, and a small number is 2 or more. For those corresponding to more than 2, the Jaccard index can be used to analyze the correlation.

[0249] Fig.11The results of the correlation analysis are shown. The Jaccard index, also known as the Jaccardsimilarity coefficient, is used to compare the similarities and differences between finite sample sets. The larger the Jaccard coefficient value, the higher the sample similarity. Based on the analysis, the correlation between a series of barcode pairs (cell label pairs) can be obtained. It can be seen that some beads have a high correlation, indicating potential multi-cellular droplets. Most single-cell droplet beads have the lowest correlation.

[0250] Fig.12 It is the result of cell barcode pairing analysis. Fig.12 The middle rows and columns are barcodes, and the darker the color, the higher the correlation. Line segments connected together are shown to potentially come from the same droplet.

[0251] Example 2

[0252] Example 2 provides a method for preparing a sequencing library and performing high-throughput sequencing. The provided method is obtained through several steps, including: preparation of label carrier microbeads, preparation of single cell suspension, preparation of microbeads, droplet generation, demulsification, reverse transcription RT reaction, enzyme digestion, cDNA amplification, fragmentation enzyme library construction, high-throughput sequencing, etc.

[0253] 1. Preparation of microbead carriers with droplet tag sequences

[0254] 1.1 Incubation of oligonucleotide coupling on microbead carrier with droplet tag sequence:

[0255] 200 pmol of modified droplet tag sequence oligonucleotides were taken from each group and mixed with 1 mg Dynabeads TM The M-280 Streptavidin magnetic beads were incubated at room temperature for 2 hours. After incubation, the magnetic beads were combined with a magnetic stand, washed 3 times, and stored in a low-salt buffer. The buffer for incubation and washing of the magnetic beads was (B&W) Buffer (1X) (5mM Tris-HCl (pH 7.5), 0.5mM EDTA, 1M NaCl)

[0256] The droplet tag sequence oligonucleotides used are:

[0257]

[0258] In the above sequence, the single underline is the PCR adapter sequence, and the following 10 bases represent the droplet tag sequence. In this embodiment, a total of 12 types are used. The wavy lines are marked with random bases, which can optimize the sequencing quality of the tag region; the base B following the random base is a degenerate base, which is G, C or T. The double underline is a polyA sequence, which is used to be captured by the oligonucleotide on another magnetic bead in the droplet (i.e., the magnetic bead carrying the cell tag sequence). The 5' end of this oligonucleotide chain is modified with Biotin and disulfide bonds in turn.

[0259] 1.2 Droplet label carrier microbead mixing and counting

[0260] After 12 groups of label carrier microbeads were prepared by incubation according to the above steps, equal amounts of magnetic beads were taken from each group and mixed. The mixed label carrier microbeads were counted for the last time to count their microbead concentrations.

[0261] 2. Single Cell Suspension Preparation

[0262] 2.1 For 293T and NIH3T3 cell lines, prepare single cell suspension by trypsin digestion, wash 1-2 times with PBS (Cat. No.: 10010031) (containing 0.04% BSA), and filter using a 40 μm cell sieve.

[0263] 2.2 Use a cell counting plate or a cell counter to detect the cell concentration.

[0264] 2.3 Depending on the cell concentration, aspirate 20,000 cells each time, collect the cell pellet by centrifugation at 300-500g, and add 100μl Cell Resuspension Buffer to resuspend the cells.

[0265] 2.4 Before loading, add 2.2 million mixed label carrier microbeads obtained in step 1.2 above to the Cell Resuspension Buffer. The specific volume is determined according to the concentration. Place on ice and wait for loading.

[0266] 3. Microbead preparation

[0267] 3.1 Refer to the magnetic bead preparation method of stLFR published by BGI (Efficient and unique co-barcoding of second-generation sequencing reads from long DNA molecules enabling cost effective and accurate sequencing, haplotyping, and de novoassembly, https: / / genome.cshlp.org / content / early / 2019 / 04 / 02 / gr.245126.118) to embed the surface oligonucleotides of magnetic beads (purchased from Spherotech, USA, catalog number SVM-200-4, https: / / www.spherotech.com / coa_mag_par.htm) to obtain magnetic beads with oligonucleotides on the surface. Pipette 200μl (220,000) of the magnetic beads into a 0.2ml PCR tube, place it on a magnetic stand for 2 minutes, and remove the supernatant.

[0268] 3.2 Remove the PCR tube from the magnetic stand and add 200 μl 1x Buffer D (1 mM EDTA, 9 mg / ml 85% KOH) to suspend the magnetic beads.

[0269] 3.3 Incubate at room temperature for 5 minutes.

[0270] 3.4 Place on the magnetic rack and let stand for 2 minutes, then remove the supernatant.

[0271] 3.5 Keep the PCR tube on the magnetic stand, add 200 μl 1x Buffer D, let stand for 30 seconds and remove the supernatant.

[0272] 3.6 Add 200 μl LSWB, let stand for 30 seconds, and then remove the supernatant.

[0273] 3.7 Repeat the previous step.

[0274] 3.8 Add 200μl Lysis Buffer (6% Ficoll PM-400 (Cat. No.: 17-0300-10), 0.2% Sarkosyl (Cat. No.: L7414), 20mM EDTA, 200mM Tris pH 7.5 (Cat. No.: T2944-1L), H2O), let stand for 30s, and then remove the supernatant. Remove the PCR tube from the magnetic stand, add 99μl Lysis Buffer and 11μl 1M DTT, so that the final concentration of DTT in this buffer is 100mM. Resuspend the magnetic beads with a low-adsorption pipette tip and place on ice.

[0275] 4. Droplet generation

[0276] 4.1 Remove the protective film on the chip surface and place it in the chip slot area of ​​the droplet generator. Figure 4 shown.

[0277] 4.2 Insert the A end of the connecting tube on the collection cover (the connecting tube that touches the bottom of the collection tube) into the Outlet hole of the chip.

[0278] 4.3 Place the 50ml syringe on the fixed stand and adjust the push rod to the initial position. Use a flat needle to connect the syringe to the connecting tube B end on the collection tube cover (the connecting tube that does not touch the bottom of the collection tube).

[0279] 4.4 Add 200 μl of droplet generation oil to the collection tube, tighten the collection cap, and place the collection tube vertically on the fixed stand.

[0280] 4.5 Use a pipette to gently blow and mix the cells, and add 100 μl of the single-cell suspension (obtained in step 2 above) to the cells well of the chip, making sure the pipette tip touches the bottom of the well.

[0281] 4.6 Use a pipette to gently blow and mix the magnetic beads, and add 100 μl of magnetic beads (obtained in step 3 above) to the beads well of the chip, making sure the pipette tip touches the bottom of the well.

[0282] 4.7 Immediately add 350 μl of droplet generation oil to the Oil well of the chip.

[0283] 4.8 Quickly pull the push rod of the syringe to the slot position and lock the push rod in the slot.

[0284] 4.9 Start the timer for 8 minutes and collect the droplets.

[0285] 4.10 After 8 minutes, immediately loosen the collection cap on the collection tube, pull out the connecting tube from the chip outlet hole, stretch the connecting tube vertically to allow the droplets in the tube to flow into the collection tube, and then replace the ordinary collection tube cap.

[0286] 4.11 Let stand at room temperature for 20 minutes to allow the mRNA molecules to fully bind to the magnetic beads.

[0287] 5. Demulsification

[0288] 5.1 Prepare the demulsification reagent by adding 10 ml 6X SSC (Cat. No.: 15557-036) and 200 μl PFO (Cat. No.: 370533-25G) into a 15 ml centrifuge tube.

[0289] 5.2 Connect the filter device to the vacuum pump, adjust the pressure parameter to 0.01MPa or 100mbar, and start the vacuum pump.

[0290] 5.3 Add 20 ml of 6X SSC to pre-treat the device.

[0291] 5.4 When there is no liquid left on the filter membrane, pour all the liquid in the collection tube evenly onto the surface of the filter membrane, and wash the collection tube twice with 2ml 6X SSC, and pour the washing solution into the filtration device.

[0292] 5.5 Mix 10 ml of demulsification reagent by inverting vigorously and quickly pour it into the filter device.

[0293] 5.6 When there is no liquid residue on the filter membrane, add 30 ml of 6X SSC to wash the magnetic beads.

[0294] 5.7 When there is no liquid residue on the filter membrane, turn off the vacuum pump and disconnect the vacuum pump from the filtration device.

[0295] 5.8 Seal the filter port of the filter device with a syringe or rubber stopper.

[0296] 5.9 Use a pipette to add 1.0 ml of collection buffer and gently blow over the entire filter surface about 20 times to suspend the magnetic beads.

[0297] 5.10 Transfer the collected solution containing magnetic beads to a 1.5 ml low-binding centrifuge tube.

[0298] 5.11 Use 1.0 ml of collection buffer to gently blow over the entire filter membrane surface about 10 times to suspend the remaining magnetic beads.

[0299] 5.12 Transfer the collected solution containing magnetic beads to a 1.5 ml low-adsorption centrifuge tube, place it on a magnetic rack, let it stand for 2 minutes, and slowly remove the supernatant.

[0300] 5.13 Remove the centrifuge tubes from the magnetic stand, use 100 μl of collection buffer to suspend the magnetic beads adsorbed on one side of the two centrifuge tubes in turn, and transfer the liquid to a 0.2 ml low-adsorption PCR tube.

[0301] 5.14 Use 100 μl of collection buffer again to suspend the magnetic beads adsorbed on one side of the two centrifuge tubes in turn, and transfer the liquid to the above 0.2 ml low-adsorption PCR tube.

[0302] 5.15 Place the PCR tube containing magnetic beads on the magnetic rack, let it stand for 2 minutes, and remove the supernatant.

[0303] 5.16 Keep the magnetic beads adsorbed, add 200 μl 6X SSC, let stand for 30 seconds, and remove the supernatant.

[0304] 5.17 Add 200 μl 5X FS Buffer, let stand for 30 seconds, and slowly remove the supernatant to avoid aspirating the magnetic beads.

[0305] 6. Reverse transcription reaction

[0306] 6.1 Prepare the reverse transcription reaction system on ice: 5μl H2O, 20μl 5x First-Strand Buffer (Cat. No.: 01E022MS), 20μl 5M Betaine (Cat. No.: B0300-1VL), 10μl 10mM dNTPs (Cat. No.: N0447L), 7.5μl 100mM MgCl2 (Cat. No.: 20-303), 5μl 50μM Template switch oligo, 5μl 100mM DTT (Cat. No.: 01E022MS), 5μl 200U / μl Alpha reverse transcriptase (Cat. No.: 01E022MS), 2.5μl 40U / μl RNase inhibitor (Cat. No.: 01E019MS).

[0307] The Alpha reverse transcriptase is an engineered MMLV reverse transcriptase, which can recognize ssDNA as a template for complementary synthesis.

[0308] 6.2 Pipette 100 μl of reverse transcription reaction system, add it to the PCR tube containing magnetic beads, and mix by pipetting.

[0309] 6.3 Perform reverse transcription reaction under the following conditions: 42°C, 90 min; 10 cycles (50°C, 2 min; 42°C, 2 min), with the hot cover set to 75°C. Due to the sedimentation of magnetic beads, flick the beads to mix them every 20 min, centrifuge briefly and continue the reaction.

[0310] 6.4 After the reaction is completed, centrifuge briefly, place on a magnetic rack, let stand for 2 minutes, and remove the reaction solution.

[0311] 6.5 Remove the PCR tube from the magnetic rack, add 200 μl TE-SDS, shake and mix, and terminate the reaction.

[0312] 6.6 After a brief centrifugation, place on a magnetic rack, let stand for 2 minutes, and remove the liquid.

[0313] 6.7 Keep the magnetic beads adsorbed, add 200 μl TE-TW, let stand for 30 seconds, and remove the supernatant.

[0314] 6.8 Repeat the above steps.

[0315] 6.9 Keep the magnetic beads adsorbed, add 200 μl 10 mM Tris pH 8.0, let stand for 30 seconds and remove the supernatant.

[0316] 7. Enzyme digestion of empty oligos that do not capture mRNA molecules on the surface of microbeads

[0317] 7.1 Enzyme digestion reaction system: 170μl H2O, 20μl 10x EXO I Buffer, 10μl EXO I enzyme.

[0318] 7.2 Pipette 200 μl of enzyme digestion reaction system, add it to the PCR tube containing magnetic beads, and vortex to mix.

[0319] 7.3 After a brief centrifugation, place in a PCR instrument and incubate at 37°C for 45 minutes with the heated lid set to 75°C. Gently flick the beads every 15 minutes to mix them well, briefly centrifuge and continue the reaction.

[0320] 7.4 After the reaction is completed, centrifuge briefly, place on a magnetic rack, let stand for 2 minutes, and remove the reaction solution.

[0321] 7.5 Remove the PCR tube from the magnetic stand, add 200 μl TE-SDS, shake and mix, and terminate the reaction.

[0322] 7.6 After a brief centrifugation, place on a magnetic rack, let stand for 2 minutes, and remove the liquid.

[0323] 7.7 Keep the magnetic beads adsorbed, add 200 μl TE-TW, let stand for 30 seconds, and remove the supernatant.

[0324] 7.8 Remove the PCR tube from the magnetic stand and add 200 μl TE-TW to suspend the magnetic beads.

[0325] 7.9 After a brief centrifugation, place on a magnetic rack, let stand for 2 minutes, and remove the liquid.

[0326] 7.10 Keep the magnetic beads adsorbed, add 200 μl H2O, let stand for 30 seconds, and remove the supernatant.

[0327] 8. cDNA Amplification

[0328] 8.1 Prepare PCR reaction system: 46 μl H2O, 4 μl 10 μM Tn Primer, 50 μl 2x KAPA HiFi Hotstart Ready mix (Cat. No. KK2602).

[0329] 8.2 Carry out PCR reaction under the following conditions: 95°C, 3 min; 13-20 cycles (98°C, 20 s; 58°C, 20 s; 72°C, 3 min).

[0330] 8.3 After PCR, use 60 μl (0.6X) AMPure XP Beads (Cat. No.: A63881) (equilibrated at room temperature for 30 min in advance) to purify and recover the PCR product. Use 200 μl (2X) AMPure XP Beads to recover the oligo fragment product in the supernatant and use Qubit dsDNA HS Kit (Cat. No.: Q32854) to detect the concentration.

[0331] 8.4 Prepare the PCR reaction system for secondary amplification of the droplet molecular tag purification product: 11μl H2O, 2μl 10μM V4-Phos-Tn-C Primer, 2μl 10μM V2-N7-index-n Primer, 25μl 2x KAPA HiFi HotstartReady mix, 10μl oligo small fragment purification product in step 8.3.

[0332] 8.5 Carry out PCR reaction under the following conditions: 95°C, 3 min; 6-10 cycles (98°C, 20 s; 58°C, 20 s; 72°C, 15 s).

[0333] 8.6 Use 200 μl (2X) AMPure XP Beads to recover the oligo secondary amplification product and detect the concentration using Qubit dsDNA HSKit.

[0334] 9. Fragmentation enzyme library construction

[0335] 9.1 For the cDNA product, perform the following library construction process: remove the fragmentase, shake and mix for 5 seconds, centrifuge briefly, and place on ice for later use.

[0336] 9.2 Take 50-300ng of the cDNA to be sheared and put it into a new 0.2ml PCR tube. The volume should be ≤16μl. The part less than 16μl should be supplemented with H2O.

[0337] 9.3 Use a pipette to draw 4.0 μl of the prepared fragmentation reaction solution (2 μl fragmentase (Cat. No.: M0348L), 2 μl 10x fragmentase buffer (Cat. No.: B0349)) into the cDNA tube, vortex for 3 times, 3 seconds each time, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0338] 9.4 Place the PCR tube on the PCR instrument and incubate at 37℃ for 10 minutes.

[0339] 9.5 Add 30 μl 0.1 M EDTA to the PCR tube, vortex to mix, and terminate the reaction.

[0340] 9.6 Centrifuge briefly to collect the reaction solution at the bottom of the tube and place it on ice.

[0341] 9.7 Use 0.6x+0.4x (i.e. 30μl+20μl) AMPure XP Beads to select the fragments of the fragmented products and use 42μl H2O to elute the DNA.

[0342] 9.8 Prepare end repair reaction solution on ice: 2.3μl H2O, 5μl 10x PNK buffer (Cat. No.: B9040L), 1.2μl 5:1 dATP:dNTP mix, 0.6μl 10U / μl T4 Polynucleotide Kinase (Cat. No.: Y9040L), 0.6μl 3U / μl T4 DNA polymerase (Cat. No.: P7080L), 0.2μl 5U / μl rTaq (Cat. No.: R500Z), 0.1μl 5U / μl Klenow fragment (Cat. No.: P7060L).

[0343] 9.9 Use a pipette to draw 10 μl of the end repair reaction solution and add it to the screened fragmentation product. Vortex to mix well and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0344] 9.10 Place the PCR tube in a PCR instrument and incubate at 37°C for 30 min or 65°C for 15 min.

[0345] 9.11 After the reaction is completed, place the reaction solution on ice.

[0346] 9.12 Prepare adapter ligation reaction solution on ice: 3.6μl H2O, 3μl 10x PNK buffer, 5μl 10μM Adapters mix, 0.8μl 100mM ATP (Cat. No.: R1441), 16μl 50% PEG 8000 (Cat. No.: EB-0.5P8K-250), 1.6μl 600U / μl T4 DNA Ligase (Cat. No.: BGE004).

[0347] 9.13 Use a pipette to slowly draw up 30 μl of the prepared adapter ligation reaction solution, add it to the end repair product, vortex to mix, and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0348] 9.14 Place the PCR tube on the PCR instrument and incubate at 23℃ for 60 minutes.

[0349] 9.15 After the reaction is completed, add 20 μl TE Buffer to make the total sample volume reach 100 μl.

[0350] 9.16 Use 50 μl AMPure XP Beads to purify and recover the ligation product, and use 48 μl H2O to elute the ligation product.

[0351] 9.17 Add 2 μl 10 μM PCR Primer and 50 μl 2x KAPA HiFi Hotstart Ready mix and vortex to mix.

[0352] 9.18 Carry out PCR reaction according to the following conditions: 95°C, 3 min; 11 cycles (98°C, 20 s; 58°C, 20 s; 72°C, 30 s).

[0353] 9.19 After PCR, use 0.6x+0.2x (i.e. 60μl+20μl) AMPure XP Beads to screen the PCR products, elute the DNA with 42μl TE Buffer, and detect the concentration using Qubit dsDNA HS Kit.

[0354] 10. High-throughput sequencing

[0355] 10.1 Take a total of 200-400ng of library into a new 0.2ml PCR tube, where the pooling ratio of the cDNA sheared PCR product and the oligo secondary PCR product of the same sample is 9:1 (you can also circularize them separately and then pool them). Use TEBuffer to make up to a total volume of 47μl.

[0356] 10.2 Add 3.0 μl 20 μM Splint Oligo primer, mix well and centrifuge briefly to the bottom of the tube.

[0357] 10.3 Place in PCR instrument and incubate at 95℃ for 3 minutes.

[0358] 10.4 After the reaction is completed, immediately transfer the PCR tube to ice and let it stand for 5 minutes.

[0359] 10.5 Prepare single-strand circularization reaction solution on ice: 3.2μl TE Buffer, 6μl 10x TA buffer, 0.6μl 100mM ATP, 0.2μl 600U / μl T4 DNA Ligase.

[0360] 10.6 Use a pipette to draw 10 μl of the prepared single-stranded circularization reaction solution and add it to the above PCR tube. Vortex to mix well and centrifuge briefly to collect the reaction solution at the bottom of the tube.

[0361] 10.7 Place the PCR tube on the PCR instrument and incubate at 37℃ for 30 minutes.

[0362] 10.8 Prepare the enzyme digestion reaction solution on ice: 1.0μl TE Buffer, 0.4μl 10x TA buffer, 1.95μl 20U / μl EXO I, 0.65μl 100U / μl EXO III.

[0363] 10.9 Pipette 4 μl of the enzyme digestion reaction solution and add it to the single-stranded cyclization product, vortex to mix, and centrifuge briefly to collect the reaction solution to the bottom of the tube.

[0364] 10.10 Place the tube on a PCR instrument and incubate at 37°C for 30 minutes.

[0365] 10.11 After the reaction is completed, add 3 μl 0.5 M EDTA, mix well and centrifuge.

[0366] 10.12 Use 90 μl PEG32 magnetic beads to recover the above enzyme digestion products, use 32 μl TE Buffer to elute the DNA, and use ssDNA Assay Kit detects the concentration of single-stranded circular library.

[0367] 10.13 The qualified libraries were sequenced using a sequencing machine with the sequencing parameters being 41+100+10.

[0368] The quality inspection results of the experimental products and libraries are as follows:

[0369] Fig.13 The quality inspection result of the obtained cDNA product is shown in FIG. Fig.14 The figure shows the quality inspection results of the oligo amplification products containing the droplet tag sequences.

[0370] After sequencing analysis, the results are shown in Table 2 and Fig.15 As shown:

[0371] Table 2 Transcriptome sequencing results

[0372]

[0373] Table 2 and Fig.15 The results of analyzing the number of cells and the number of genes in a single cell based on the transcriptome sequencing results are shown. Fig.15 Here UB represents the number of UMI counts, and GN represents the number of genes.

[0374] Fig.16The number of molecules of the droplet tag sequence captured for each combination of cell tag sequence and droplet tag sequence, that is, the number of droplet tag sequences captured by each cell tag sequence. The results shown show that most of them are captured at the level of several hundred, which means that the droplet tag sequence can be effectively dissociated from the magnetic beads and captured and labeled by the magnetic beads with cell tag sequences, and the effective number captured supports subsequent analysis.

[0375] Fig.17 The number of droplet label sequence types corresponding to each cell label sequence is displayed. Each cell label sequence corresponds to 2-8 droplet label sequences. When there are enough types of droplet label sequences, the cell label sequences can be grouped by the type combination of droplet label sequences captured by magnetic beads with cell label sequences, and the magnetic beads with cell label sequences from the same droplet can be determined by correlation calculation.

[0376] Fig.18 The number of cell label sequence types corresponding to each droplet label sequence is shown. From this figure, it can be seen that the number of cell label types corresponding to each droplet label sequence is large because there are 12 types of droplet label sequences in this embodiment, and the number of cell label sequences is more than one thousand. After increasing the types of droplet label sequences, this parameter is reduced to increase the discrimination of cell label sequences.

[0377] Fig.19 The results of the correlation analysis are shown. The Jaccard index, also known as the Jaccardsimilarity coefficient, is used to compare the similarities and differences between finite sample sets. The larger the Jaccard coefficient value, the higher the sample similarity. Based on the analysis, the correlation between a series of barcode pairs can be obtained, and it can be seen that the correlation of some cell label sequences calculated based on the droplet label sequence is higher.

[0378] Fig. 20 The results of the cell barcode pairing analysis, where both rows and columns are barcodes, and the darker the color, the higher the correlation. Lines connected together are shown to potentially come from the same droplet.

[0379] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine the different embodiments or examples described in this specification and the features of the different embodiments or examples, without contradiction.

[0380] Although the embodiments of the present invention have been shown and described above, it is to be understood that the above embodiments are exemplary and are not to be construed as limitations of the present invention. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present invention.

Claims

1. A droplet, characterized in that: contain: Biological material, the biological material containing nucleic acid molecules; a droplet identification molecule carrying a droplet tag sequence; and A first vector, wherein the first vector carries a capture sequence and a cell label sequence, Wherein, the capture sequence is used to capture at least one of the nucleic acid molecule and the droplet identification molecule; The droplet identification molecule is provided in the form of a second carrier, and the second carrier carries a plurality of droplet identification molecules; The droplet identification molecule is connected to the second carrier via a covalent bond, and the droplet identification molecule further includes a recognition sequence of a capture sequence, and the recognition sequence of the capture sequence is connected to the droplet tag sequence.

2. The droplet according to claim 1, characterized in that The nucleic acid molecule is mRNA or DNA.

3. The droplet according to claim 1, characterized in that The biological material is provided in the form of cells.

4. The droplet according to claim 3, characterized in that Each of the droplets contains one of the cells, and each of the droplets contains at least one of the vectors.

5. The droplet according to claim 1, characterized in that The droplet identification molecule further includes a recognition sequence of a capture sequence, and the recognition sequence of the capture sequence is connected to the droplet tag sequence.

6. The droplet according to claim 1, characterized in that Further comprising: a cell lysis reagent.

7. The droplet according to claim 1, characterized in that The covalent bond is a disulfide bond.

8. The droplet according to claim 1, characterized in that The droplet further comprises: a cleavage reagent, which can destroy the connection between the plurality of droplet identification molecules and the second carrier.

9. The droplet according to claim 8, characterized in that The cleavage reagent is DTT.

10. The droplet according to claim 1, characterized in that The droplets are water-in-oil structures.

11. A suspension, characterized in that The method comprises a plurality of droplets according to any one of claims 1 to 10, wherein, for at least two droplets, the droplet tag sequences have different nucleic acid sequences.

12. A method for analyzing single cell nucleic acid, characterized in that: include: Providing a single cell suspension, the single cell suspension containing single dispersed cells, mixing the single cell suspension with a droplet identification molecule to obtain a cell suspension, the droplet identification molecule carrying a droplet tag sequence; Placing the cell suspension, the first carrier and the oil at different positions of a microfluidic chip respectively, so that the cell suspension, the first carrier and the oil pass through the same channel, so as to obtain droplets, wherein the droplets are the droplets according to any one of claims 1 to 9; Performing demulsification treatment on the droplets and building a library to obtain a sequencing library; The sequencing library is subjected to sequencing analysis to obtain the nucleic acid information of the single cell.

13. The method according to claim 12, characterized in that The concentration of the single cell suspension is 100 to 200 cells per microliter, and the concentration of the droplet identification molecule is 10 5 ~10 8 The droplet identification molecule has a copy number.

14. The method according to claim 12, characterized in that The concentration of the vector is 2000 to 3000 vectors per microliter.

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