Non-instrument-dependent high-throughput single cell library building method

Abstract

The invention relates to the field of molecular biological technology high-throughput single cell sequencing, and discloses a non-instrument-dependent high-throughput single cell library building method which comprises the following steps: S1, water-in-oil microdroplet generation; s2, temperature-controlled cell lysis: heating the water-in-oil system to activate the lysis reagent for cell lysis; s3, demulsification and microsphere cleaning: adding a demulsification buffer solution and perfluorooctanol for demulsification, and cleaning to remove invalid mRNA; s4, molecular tag release and reverse transcription; s5, amplifying and purifying the cDNA; and S6, library construction and sequencing. According to the method for generating the water-in-oil droplets without depending on microfluidic equipment, the water-in-oil droplets are generated, about 1-10% of effective droplets contain cells, molecular bar code gel beads and a cell lysis system at the same time, the polycellular rate in the effective droplets is lower than 5%, the cell capture rate is about 45%, and the method can replace the method for generating the water-in-oil droplets through the microfluidic equipment.

Description

Technical Field

[0001] This invention relates to the field of high-throughput single-cell sequencing in molecular biology, specifically a non-instrument-dependent high-throughput single-cell library construction method. Background Technology

[0002] Single-cell sequencing refers to sequencing technology that obtains the genetic information of a single cell. It has ushered in a new revolution at the cellular level and is a cutting-edge technology in basic life science research, widely used in many fields such as tumor microenvironment, immunotherapy, embryonic development, and the occurrence and development of neurological diseases. Currently, the mainstream single-cell sequencing technologies on the market are droplet microfluidics and microplate technology. Droplet microfluidics uses microfluidic devices to encapsulate cell suspensions and gel microspheres carrying unique molecular tag sequences in water-in-oil droplets. Each effective droplet theoretically contains one cell and one gel microsphere. The core advantages of this technology are high throughput, standardization, and automation, and it accounts for more than 80% of the application market. Microplate technology uses sample loading devices to load single cells and magnetic beads into the independent wells of a precision-machined microplate chip. This technology is characterized by high capture efficiency, low amplification bias, and flexible throughput, and is especially suitable for rare samples and multi-omics concatenation, but its throughput limit is usually lower than that of droplet technology.

[0003] Existing single-cell technologies achieve efficient and reliable single-cell sequencing through expensive and complex instruments and equipment. However, the high cost of the equipment and the limited throughput limit lead to increased sequencing costs and make it difficult to meet the needs of large-scale research. Summary of the Invention

[0004] To address the shortcomings of existing technologies, this invention provides an instrument-independent, high-throughput single-cell library preparation method, which solves the problem that the expensive and complex instruments and equipment used in existing single-cell technologies lead to increased sequencing costs and make it difficult to meet the needs of large-scale research.

[0005] To achieve the above objectives, the present invention provides the following technical solution: a non-instrument-dependent high-throughput single-cell library construction method, comprising the following steps: S1: Water-in-oil droplet generation: Based on the principle of sequential partitioning of particle template emulsification, in a low-temperature environment, cell suspension, light-triggered microspheres carrying molecular tag sequences, and temperature-controlled cell lysis reagent are mixed and then oil is added to generate droplets. Water-in-oil droplets are generated through oscillation. S2: Temperature-controlled cell lysis: The water-in-oil system is heated to activate the lysis reagent and induce cell lysis. S3: Demulsification and microsphere washing: Demulsification buffer and perfluorooctyl alcohol were added to break the emulsion, and washing was performed to remove ineffective mRNA; S4: Molecular tag release and reverse transcription: Microbeads are stimulated by specific light intensity to release molecular tag sequences and the mRNA they capture, and cDNA is synthesized in an optimized reverse transcription system; S5: cDNA amplification and purification: cDNA is amplified using a PCR amplification system, and the product is purified using magnetic beads; S6: Library construction and sequencing: Construct single-cell libraries using a library construction kit and perform sequencing.

[0006] Cell suspension was prepared by resuspending cells in DPBS solution containing 0.04% BSA and 1% Pluronic F127 using the above-mentioned technique, and adjusting the concentration to 4-6 × 10⁻⁶. 6Cells / ml range; take 5 μL of suspension containing 20,000 to 30,000 cells and mix with 5 μL of 20 U / μL SUPERase InRNase Inhibitor. Add 50 μL of light-triggered gel microspheres, 9.38 μL of 800 U / mL proteinase K, 3.5 μL of 1 MDTT, 2.5 μL of 1×Plusion HF Buffer, 5 μL of 0.2% BSA, and 19.62 μL of nuclease-free water to a pre-chilled sample tube. Mix well with the cell suspension and add 200 μL of pre-chilled droplet oil. Shake horizontally at 3000 rpm for 10 seconds and vertically for 1 minute on a desktop shaker, with the sidewall of the sample tube lightly touching the edge of the rotor. Let stand for 1 minute. After centrifugation, remove 90% of the bottom oil phase, transfer the water-in-oil sample to an incubation container, lyse at 50°C for 30 minutes, and inactivate proteinase K at 80°C for 10 minutes. Add 180 μL of demulsification buffer and 40 μL of perfluorooctyl alcohol, wherein the demulsification buffer contains 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, and 50 mM DTT, and mix by inversion until demulsification is complete. After brief centrifugation, remove the lower oil phase, transfer the retained solution to a new tube, and wash with 1 mL of washing buffer containing 100 mM Tris-HCl, 150 mM KCl, 6 mM MgCl2, 20 mM DTT, and 1% Pol. Wash the microspheres three times with oxamer188, resuspend the precipitate in 50 μL for later use; add 50 μL of reverse transcription mixture containing 2% Ficoll PM400, 2 μM MTO, 1 mM dNTPs, 1 U / μL LR Nase inhibitor, 1 U / μL Maxima HMinus reverse transcriptase and 1× RT-Buffer; after UV release of the molecular tag, execute the reverse transcription program: 25°C / 5 min, 42°C / 90 min, 85°C / 10 min; aspirate the supernatant, add 120 μL of washing buffer to the microsphere tube, mix well and centrifuge, combine the supernatants and purify with 1.5× DNA magnetic beads, 4 Elute with 6 μL LEB; add 54 μL cDNA amplification mixture containing 50 μL KAPAHiFiHotStartReadyMixPCR reagent and 4 μL 10 μM cDNA primers, and perform PCR program: initial denaturation at 95°C for 3 min; 20 cycles of denaturation at 98°C for 15 s, annealing at 67°C for 20 s, and extension at 72°C for 3 min; final extension at 72°C for 5 min; purify the amplified product twice with 0.6× DNA magnetic beads, elute with 40 μL LEB, construct a library using NexteraXT DNA library preparation kit, and sequence using Illumina platform.

[0007] A method for generating water-in-oil droplets without relying on microfluidic devices is proposed. Based on the principle of sequential partitioning of particle template emulsification, barcode microspheres are used as particle templates. The barcode microspheres, cell suspension, cell lysis reagent, and droplet-generating oil are sequentially partitioned through oscillation to generate water-in-oil droplets. Approximately 1-10% of the effective droplets simultaneously contain cells, molecular barcode gel beads, and cell lysis systems. Moreover, the multicellularity rate of the effective droplets is less than 5%, and the cell capture rate is approximately 45%. This method can replace the generation of water-in-oil droplets through microfluidic devices.

[0008] Preferably, in S1, the cell suspension is resuspended in a DPBS solution containing 0.04%~0.1% BSA and 0.1%~1% Pluronic F127; and the final concentration of the temperature-controlled cell lysis reagent includes: 30~300 U / ml proteinase K, 20~70 mMDTT, 0.01%~0.1% BSA and 1%~10% Plusion® HF Buffer.

[0009] Preferably, in S1, the number of light-triggered microspheres is more than 10 times the number of cells; the volume of droplet-generated oil is more than 1.5 times the volume of aqueous phase; the oscillation operation uses a desktop shaker at a maximum speed of 3000 rpm, first oscillating horizontally for 10 seconds, then oscillating vertically for 1-2 minutes, with the edge of the shaker rotor lightly touching the side wall of the centrifuge tube.

[0010] Preferably, the phototriggered microspheres in S1 are magnetic beads or gel microspheres carrying molecular tag sequences, which are linked by photosensitive chemical bonds and include R1 sequencing primers, cell barcodes, UMI and mRNA capture sequences.

[0011] Preferably, the temperature-controlled lysis procedure in S2 is as follows: cell lysis is performed by incubation at 37℃~60℃ for 10~30 minutes, followed by incubation at 80℃~85℃ for 5~10 minutes to inactivate proteinase K; all operations before S2 lysis are performed in a low-temperature environment of ≤18℃.

[0012] Preferably, the demulsification buffer components in S3 are: 250mM Tris-HCl, 375mM KCl, 15mM MgCl2, and 50mM DTT; while perfluorooctyl alcohol is added for demulsification, and the mixture is inverted and mixed 10-20 times.

[0013] Preferably, the washing buffer components in S3 are: 100 mM Tris-HCl, 150 mM KCl, 6 mM MgCl2, 20 mM DTT and 1% Poloxamer™ 188. The microsphere washing is repeated 3 to 4 times by resuspension, centrifugation and discarding of supernatant. All centrifugation steps are performed by turning off the power and pausing the centrifugation. After a short centrifugation, the microspheres are allowed to stand and separate into layers.

[0014] Preferably, the reverse transcription system in S4 contains: 2%~4% Ficoll PM400, 1~5 μM MTO, 0.5~1 mM dNTPs, 0.5~2 U / μL LRNase inhibitor, 1~10 U / μL Maxima HMinus reverse transcriptase and 1×RT Buffer; the reverse transcription program in S4 is: 25℃ / 5 min, 42℃ / 90 min and 85℃ / 10 min; the molecular tag release in S4 is achieved by breaking the photosensitive chemical bonds through ultraviolet irradiation, releasing the molecular tag sequence on the microspheres and the mRNA it captures.

[0015] Preferably, cDNA amplification in S5 uses the KAPA HiFi HotStart Ready Mix PCR Kit. The amplification program is as follows: first, initial denaturation is performed at 95℃ for 3 min; then, the operation is repeated 20 times. Each cycle includes: DNA denaturation at 98℃ for 15 s, primer annealing at 67℃ for 20 s, DNA extension at 72℃ for 3 min, and final extension at 72℃ for 5 min. Purification in S5 is performed twice using 0.6~0.8× DNA magnetic beads.

[0016] Preferably, the S6 Chinese library is constructed using the NexteraXT DNA library construction kit, along with primers that match the microsphere molecular tag.

[0017] This invention provides an instrument-independent, high-throughput single-cell library construction method. It has the following beneficial effects: 1. This invention provides a method for generating water-in-oil droplets without relying on microfluidic devices. Based on the principle of sequential partitioning in particle template emulsification, barcode microspheres are used as particle templates. The barcode microspheres, cell suspension, cell lysis reagent, and droplet-generating oil are sequentially partitioned through oscillation to generate water-in-oil droplets. Approximately 1-10% of the effective droplets simultaneously contain cells, molecular barcode gel beads, and a cell lysis system. Furthermore, the multicellularity rate in the effective droplets is less than 5%, and the cell capture rate is approximately 45%. This method can replace the generation of water-in-oil droplets using microfluidic devices.

[0018] 2. This invention provides a temperature-controlled cell lysis method to replace the conventional high-throughput single-cell Triton X-100 lysis system. In this temperature-controlled method, after cells and the lysis reagent are mixed at low temperatures, the lysis reagent is inhibited. After the formation of water-in-oil droplets, the cell lysis reagent is activated by heating, thus solving the problem that cell suspension and cell lysis reagent cannot coexist in the same container. 3. This invention provides a cell resuspension suitable for temperature-controlled cell lysis methods, which has the effects of improving cell dispersion, stabilizing enzyme activity, protecting nucleic acids and reducing nucleic acid adsorption.

[0019] 4. This invention optimizes the reverse transcription system by using 2% to 4% Ficoll PM400 to replace commonly used macromolecular crowding agents such as betaine, PEG8000, or a combination of PEG8000 and Ficoll PM400, thereby improving reverse transcription efficiency and increasing cDNA yield by 40%.

[0020] 5. This invention generates water-in-oil droplets through oscillation and provides a matching high-throughput single-cell transcription library construction method: optimized cell resuspension, temperature-controlled cell lysis system, optimized high-efficiency reverse transcription system, and combined with KAPAHiFiHotStartReadyMixPCRKit and NexteraXTDNAlibrarypreparationkit to complete single-cell library construction. This method has a cell capture rate of approximately 45%, a multi-cell rate of less than 5%, and a multi-bead rate of less than 1%. Compared with microfluidic single-cell libraries, the identified cell types are consistent, and the similarity of cell type distribution and cell gene type number is greater than 80%. Attached Figure Description

[0021] Figure 1 This is a process flow diagram of a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention; Figure 2 This is a peak control diagram of cDNA products obtained by different cell resuspension formulations in a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention; Figure 3 Box plot of cDNA yield variation obtained by different macromolecular crowding reagents in the reverse transcription system of a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention; Figure 4 This is a microscopic image of water-in-oil droplets detected by a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention. Figure 5 This is a cDNA peak diagram of a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention; Figure 6 Violin plot of gene species count for a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention; Figure 7 This is a schematic diagram illustrating the differences in cell clusters in a non-instrument-dependent high-throughput single-cell library construction method proposed in this invention. Detailed Implementation

[0022] The technical solution of the present invention will now be clearly and completely described with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0023] Please see the appendix Figure 1 - Appendix Figure 7 This invention provides a non-instrument-dependent high-throughput single-cell library construction method, comprising the following steps: S1: Water-in-oil droplet generation: Based on the principle of sequential partitioning of particle template emulsification, in a low-temperature environment, cell suspension, light-triggered microspheres carrying molecular tag sequences, and temperature-controlled cell lysis reagent are mixed and then oil is added to generate droplets. Water-in-oil droplets are generated through oscillation. S2: Temperature-controlled cell lysis: The water-in-oil system is heated to activate the lysis reagent and induce cell lysis. S3: Demulsification and microsphere washing: Demulsification buffer and perfluorooctyl alcohol were added to break the emulsion, and washing was performed to remove ineffective mRNA; S4: Molecular tag release and reverse transcription: Microspheres are stimulated by specific light intensity to release molecular tag sequences and the mRNA they capture, and cDNA synthesis is carried out in an optimized reverse transcription system; S5: cDNA amplification and purification: cDNA is amplified using a PCR amplification system, and the product is purified using magnetic beads; S6: Library construction and sequencing: Construct single-cell libraries using a library construction kit and perform sequencing.

[0024] Specifically, cell preparation and water-in-oil system generation: The prepared cell suspension was resuspended in DPBS solution containing 0.04% BSA and 1% Pluronic F127, and the concentration was adjusted to 4~6×10⁻⁶. 6 Cells / ml; Take 5 μL of suspension containing 20,000 to 30,000 cells and mix with 5 μL of 20 U / μL SUPERase InRNase Inhibitor; Prepare cell lysis mixture in ice-cold sample tubes, containing 50 μL of light-triggered gel microspheres, 9.38 μL of 800 units / ml proteinase K, 3.5 μL of 1MDTT, 2.5 μL of 1×Plusion HF Buffer, 5 μL of 0.2% BSA and 19.62 μL of nuclease-free water. Add the cell suspension and mix well, then add 200 μL of pre-cold droplets to generate oil; Use a desktop shaker to oscillate laterally for 10 seconds and longitudinally for 1 minute at the maximum speed of about 3000 rpm. During this process, the side wall of the sample tube should lightly touch the edge of the shaker rotor. After standing for 1 minute, remove 90% of the bottom oil phase.

[0025] Temperature-controlled lysis and demulsification: Transfer the water-in-oil sample tube to an incubation container and lyse at 50°C for 30 minutes, then inactivate proteinase K at 80°C for 10 minutes. Add 180 μL of demulsification buffer and 40 μL of perfluorooctanoic acid along the tube wall. The demulsification buffer contains 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, and 50 mM DTT. Invert and mix until demulsification is complete. After brief centrifugation, separate into three layers. Thoroughly remove the lower oil phase and transfer the remaining solution to a new tube. Wash the microspheres three times with 1 mL of washing buffer containing 100 mM Tris-HCl, 150 mM KCl, 6 mM MgCl2, 20 mM DTT, and 1% Poloxamer 188. Finally, resuspend the microsphere precipitate in 50 μL for later use.

[0026] Reverse transcription and purification: 50 μL of reverse transcription mixture containing 2% Ficoll PM400, 2 μM MTO, 1 mM dNTPs, 1 U / μL L N-ase inhibitor, 1 U / μL Maxima H-Minus reverse transcriptase, and 1× RT-Buffer was added to the microspheres. After the microspheres released the molecular tag sequence and the captured mRNA under specific light stimulation, a reverse transcription program was performed: 25°C for 5 minutes, 42°C for 90 minutes, and 85°C for 10 minutes. The supernatant was collected, and 120 μL of washing buffer was added to the microsphere tubes. After centrifugation, the supernatants were collected and combined, and purified using 1.5× DNA magnetic beads and eluted with 46 μL LEB.

[0027] cDNA amplification and library construction: Add 54 μL of cDNA amplification mixture containing 50 μL of KAPAHiFiHotStartReadyMixPCR reagent and 4 μL of 10 μM cDNA primers. The specific PCR procedure is as follows: First, initial denaturation is performed at 95℃ for 3 min; then, the operation is repeated 20 times, each cycle including: DNA denaturation at 98℃ for 15 s, primer annealing at 67℃ for 20 s, DNA extension at 72℃ for 3 min, and final extension at 72℃ for 5 min. The amplified product is purified twice with 0.6× DNA magnetic beads and eluted with 20 μL LEB. Take 10 μL of cDNA product and construct a library using the Nextera XT DNA library construction kit, followed by sequencing on the Illumina platform.

[0028] See appendix Figure 1In S1, the cell suspension was resuspended in DPBS containing 0.04%–0.1% BSA and 0.1%–1% Pluronic F127; and the final concentration of the temperature-controlled cell lysis reagent contained 30–300 U / ml proteinase K, 20–70 mM DTT, 0.01%–0.1% BSA and 1%–10% Plusion® HF Buffer.

[0029] Specifically, in S1, the cell suspension needs to be resuspended using Dulbecco phosphate buffer containing 0.04% to 0.1% bovine serum albumin and 0.1% to 1% Pluronic F127 surfactant. The final concentrations of the temperature-controlled cell lysis reagent system need to be precisely prepared: proteinase K at a final concentration of 30 U / ml to 300 U / ml, dithiothreitol at a final concentration of 20 mmol / L to 70 mmol / L, and bovine serum albumin at a final concentration of 0.01% to 0.1%, while adding 1% to 10% of the total volume of Plusion HF Buffer. In practice, the resuspended cell suspension, light-triggered microspheres carrying molecularly tagged sequences, and the above-mentioned temperature-controlled lysis reagent are thoroughly mixed in a pre-chilled reaction tube on ice. Then, pre-chilled droplets are added to generate a dedicated oil phase, and the stable generation of water-in-oil droplets is achieved through precisely controlled oscillation. The synergistic effect of dithiothreitol and proteinase K in this lysis reagent needs to be maintained in a completely inhibited state throughout the emulsification process, and its activity will be specifically activated in the subsequent temperature-controlled step.

[0030] See appendix Figure 1 In S1, the number of light-triggered microspheres is more than 10 times the number of cells; the volume of droplet-generated oil is more than 1.5 times the volume of aqueous phase; the oscillation operation is performed using a desktop oscillator at a maximum speed of 3000 rpm, first oscillating horizontally for 10 seconds, then oscillating vertically for 1-2 minutes, with the edge of the oscillator rotor lightly touching the side wall of the centrifuge tube.

[0031] Specifically, in S1, the number of light-triggered microspheres used must exceed the number of cells, specifically more than 10 times the total number of cells, to ensure effective pairing of single cells with microspheres. The volume of the droplet-generating oil added must be controlled to be at least 1.5 times the volume of the aqueous phase mixture to ensure emulsification. The oscillation operation is performed using a desktop shaker: first, adjust the instrument to the maximum speed of about 3000 rpm for horizontal oscillation for 10 seconds; then immediately switch to vertical oscillation mode and continue oscillating for 1 to 2 minutes. The key point of operation is that during the oscillation process, the edge of the shaker rotor should lightly touch the side wall of the centrifuge tube to transfer oscillation energy through physical collision, avoiding direct contact with the bottom or cap of the tube. This oscillation method can ensure efficient dispersion of cell suspension, light-triggered microspheres, and lysis reagent in the oil phase, forming a stable water-in-oil microdroplet structure. The entire oscillation process must be continuous.

[0032] See appendix Figure 1 In S1, the phototriggered microspheres are magnetic beads or gel microspheres carrying molecular tag sequences. The molecular tag sequences are linked by photosensitive chemical bonds and include R1 sequencing primers, cell barcodes, UMI, and mRNA capture sequences.

[0033] Specifically, in the generation of S1 water-in-oil droplets, the phototriggered microspheres used are magnetic beads carrying molecular tag sequences or gel microspheres insoluble in dithiothreitol. The core feature of these microspheres is that their molecular tag sequences are covalently linked to the microsphere matrix via photosensitive chemical bonds. This linkage can achieve specific cleavage and release under subsequent specific light intensity stimulation. The structure of the molecular tag sequence includes four essential functional regions: an R1 sequencing primer sequence for initiating extension of subsequent high-throughput sequencing; a cell barcode sequence for distinguishing nucleic acid molecules from different cell sources; a unique molecular identifier (UMI) sequence for correcting amplification bias and achieving absolute transcript quantification; and an mRNA capture sequence for specifically binding to target nucleic acids. Its specific form can be a polythymidine nucleotide chain, a template-converting oligonucleotide, or a primer sequence designed for a specific target gene. The complete structural design of this molecular tag is compatible with conventional single-cell sequencing technology, ensuring the resolvability of sequencing data.

[0034] See appendix Figure 1 The temperature-controlled lysis procedure in S2 is as follows: cell lysis is performed by incubating at 37℃~60℃ for 10~30 minutes, followed by incubation at 80℃~85℃ for 5~10 minutes to inactivate proteinase K; all operations before S2 lysis are performed in a low-temperature environment of ≤18℃.

[0035] Specifically, during the S2 temperature-controlled cell lysis process, all pre-lysis operations must maintain an ambient temperature not exceeding 18°C ​​to ensure that proteinase K is in a completely inhibited state. The lysis procedure is executed in two stages: first, the water-in-oil system is incubated at 37°C to 60°C for 10 to 30 minutes to activate proteinase K hydrolysis activity and achieve cell lysis; then, the temperature is raised to 80°C to 85°C and incubated for 5 to 10 minutes to completely inactivate proteinase K and terminate the reaction. This temperature-controlled design relies on the enzymatic properties of proteinase K—its activity is inhibited at low temperatures, while its activity increases in the range of 20°C to 60°C.

[0036] See appendix Figure 1 The demulsification buffer in S3 consists of 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, and 50 mM DTT; perfluorooctyl alcohol is added for demulsification, and the mixture is inverted and mixed 10-20 times.

[0037] Specifically, in the S3 demulsification and microsphere washing stage, a demulsification buffer solution with specific components needs to be prepared: this buffer solution contains 250 mmol / L Tris-HCl, 375 mmol / L potassium chloride, 15 mmol / L magnesium chloride, and 50 mmol / L dithiothreitol. During the demulsification process, slowly add 180 μL of the demulsification buffer and 40 μL of perfluorooctyl alcohol along the side wall of the sample tube, and manually invert and mix 10 to 20 times until the water-in-oil emulsion is completely demulsified to form a homogeneous solution. If the demulsification is incomplete and the solution is unevenly layered, add 5 to 20 μL of perfluorooctyl alcohol and repeat the inverting and mixing operation. Then, perform a brief centrifugation to separate the solution into three layers: the bottom layer is the oil phase, the middle layer is the gel microsphere aggregate layer, and the top layer is the aqueous phase. At this time, the bottom oil phase needs to be removed to avoid residual undemulsified droplets affecting the efficiency of subsequent reverse transcription reactions.

[0038] See appendix Figure 1 The washing buffer in S3 consists of 100 mM Tris-HCl, 150 mM KCl, 6 mM MgCl2, 20 mM DTT, and 1% Poloxamer™ 188. The microsphere washing is repeated 3-4 times by resuspending, centrifuging, and discarding the supernatant. All centrifugation steps are performed by turning off the power and pausing the centrifugation. After a short centrifugation, the microspheres are allowed to stand and separate into layers.

[0039] Specifically, the cleaning process for S3 microspheres requires the use of a specific cleaning buffer: this buffer consists of 100 mmol / L Tris-HCl, 150 mmol / L potassium chloride, 6 mmol / L magnesium chloride, 20 mmol / L dithiothreitol, and 1% nonionic surfactant Poloxamer™ 188. The cleaning operation is strictly performed according to the following procedure: Washing cycle: Add 1 mL of washing buffer to the microsphere precipitate each time, and gently resuspend the microspheres by pipetting. Centrifugal separation: A desktop centrifuge is used for short-term centrifugation. The centrifugation operation needs to be stopped slowly by turning off the power, that is, cutting off the centrifuge power so that the rotor can decelerate and stop naturally, avoiding damage to the microsphere structure caused by sudden stop of high-speed centrifugation. Layer removal: After centrifugation, let stand for 1 minute until the microspheres are completely settled at the bottom of the tube, and then thoroughly remove the supernatant; Repeating number of times: The above resuspension-centrifugation-discarding supernatant process is repeated 3 to 4 times to thoroughly remove any residual free mRNA and impurities outside the droplets.

[0040] This cleaning system reduces surface tension using the nonionic surfactant Poloxamer™ 188 and maintains a reducing environment using dithiothreitol, ensuring the integrity of the molecular tag sequence on the microsphere surface and providing a clean reaction interface for subsequent stimulation of the microspheres by specific light intensity to release the molecular tag sequence and the captured mRNA.

[0041] See appendix Figure 1 The reverse transcription system in S4 contains: 2%~4% Ficoll PM400, 1~5 μM MTO, 0.5~1 mM dNTPs, 0.5~2 U / μL LRNase inhibitor, 1~10 U / μL Maxima HMinus reverse transcriptase and 1×RT Buffer; the reverse transcription program in S4 is: 25℃ / 5 min, 42℃ / 90 min and 85℃ / 10 min; the molecular tag release in S4 is achieved by breaking the photosensitive chemical bonds through specific light intensity irradiation, releasing the molecular tag sequence on the microsphere and the mRNA it captures.

[0042] Specifically, the S4 molecular tag release and reverse transcription stage requires an optimized reverse transcription reaction system. Its key components and concentration ranges include: 2% to 4% of the total volume of sucrose PM400 macromolecular crowding agent, 1 mmol / L to 5 mmol / L of template-converting oligonucleotides, 0.5 mmol / L to 1 mmol / L of a mixture of deoxyribonucleotide triphosphates, 0.5 μL to 2 μL of a ribonuclease inhibitor, 1 μL to 10 μL of Maxima HMinus reverse transcriptase, and a 1-fold concentration of reverse transcription buffer. The specific operating procedure is as follows: Molecular tag release: After the cleaned microspheres are mixed with the reverse transcription system, the sample tube is irradiated with ultraviolet light of a specific wavelength, which triggers the breakage of the photosensitive chemical bonds on the surface of the microspheres, thereby releasing the covalently linked molecular tag sequence and the messenger ribonucleic acid molecules it captures. After release, it is necessary to gently blow and mix to ensure the uniformity of the reaction.

[0043] Reverse transcription procedure: A three-step temperature program was performed in a polymerase chain reaction apparatus with a 105°C heat-controlled lid—first, primer annealing was achieved by incubation at 25°C for 5 minutes, followed by synthesis of complementary deoxyribonucleic acid (DNA) chains at 42°C for 90 minutes, and finally, treatment at 85°C for 10 minutes was performed to completely inactivate the reverse transcriptase, ensuring the efficient synthesis of complementary DNA carrying molecular tags.

[0044] See appendix Figure 1 In S5, cDNA amplification was performed using the KAPA HiFi HotStart ReadyMixPCR Kit. The amplification program was as follows: first, initial denaturation was performed at 95℃ for 3 min; then, the operation was repeated 20 times, with each cycle consisting of: DNA denaturation at 98℃ for 15 s, primer annealing at 67℃ for 20 s, DNA extension at 72℃ for 3 min, and final extension at 72℃ for 5 min. Purification in S5 was performed twice using 0.6~0.8× DNA magnetic beads.

[0045] Specifically, in stage S5, the cDNA amplification step, the following procedure was performed using the KAPA HiFi HotStart Ready Mix PCR Kit in a polymerase chain reaction (PCR) system: First, an initial denaturation treatment was performed at 95°C for 3 minutes to activate the hot-start DNA polymerase in the kit and fully denature the template double-stranded DNA. This was followed by 20 cycles of amplification. In each cycle, the first step involved maintaining the temperature at 98°C for 15 seconds to achieve complete denaturation and separation of the DNA template strand; the second step involved rapidly cooling to 67°C and holding for 20 seconds to ensure precise annealing of the specific primers to the target DNA sequence; and the third step involved extension at 72°C. The reaction was carried out at 3°C ​​for 3 minutes to allow the DNA polymerase to fully exert its chain extension synthesis capability. After all 20 cycles, the final extension step was carried out at 72°C for 5 minutes to ensure that all the newly synthesized chains were fully extended. The subsequent S5 purification operation adopted the carboxyl magnetic bead method. Specifically, the amplified product was purified twice consecutively with 0.6 to 0.8 times the volume of DNA magnetic beads according to the standard operating procedure. The purpose was to efficiently remove primer dimers, DNA polymerase, residual salt ions from the DNA synthesis reaction, and other low molecular weight impurities, while also achieving the sorting and recovery of DNA fragments, and finally obtaining high-quality amplified DNA products with purity that meets the requirements of downstream applications.

[0046] See appendix Figure 1 The S6 Chinese library was constructed using the NexteraXT DNA library construction kit, along with primers that matched the microsphere molecular tags.

[0047] Specifically, in the S6 library construction stage, the NexteraXT DNA library preparation kit was used to complete the library preparation, and the library was finally recovered using 20 μL of elution buffer.

[0048] The prepared library must undergo quality verification: fragment distribution should be detected using an Agilent 2100 bioanalyzer, and the main peak range of a qualified library should be 300 to 800 base pairs; the library concentration should be higher than 2 nmol / L as determined by quantitative polymerase chain reaction; quality control sequencing should be performed on the Illumina sequencing platform, and the Q30 quality score of the library reads after data quality control should reach more than 90%, with the effective sequence ratio not less than 80%. The entire library construction process does not rely on automated equipment, which conforms to the principle of non-instrument-dependent library construction. If it is necessary to adjust the library construction scale, other commercial library construction kits with the same functions can be selected to replace the operation.

[0049] Example 1: Through repeated research, the cell resuspension formulation was screened and optimized to obtain a cell resuspension reagent formulation suitable for the proteinase K cell lysis system: DPBS containing 0.04%~0.1% BSA and 1% Pluronic F127. Adding BSA to the cell resuspension or to the cell lysis system can improve the adsorption of cDNA products.

[0050] See appendix Figure 2 This study demonstrates the effects of different cell resuspensions on cDNA products.

[0051] By comparing the peak patterns of cDNA products obtained from four cell resuspensions, the cDNA products obtained from the two cell resuspensions without added BSA showed abnormal peak patterns during Agilent 2100 quality control (HighSensitivity DNA Kit). After adding BSA, the cDNA product peak patterns returned to normal. Adding BSA reduced the cDNA product's adsorption capacity, restoring the cDNA quality control to normal. FBS cannot be used to replace BSA, as FBS affects the cleavage efficiency of proteinase K.

[0052] Example 2: Through repeated research, the inventors screened and optimized the formulation of PCR reagents for PCR amplification. The optimized reagent formulation is as follows: 2%~4% Ficoll PM400, 2.5µM MTSO, 1mM dNTPs, 1U / µl RNase inhibitor, 1U / µL Maxima HMinus Reverse Transcriptase, 50mM Tris-HCl, 75mM KCl, 3mM MgCl2 and 10mM DTT.

[0053] See appendix Figure 3 The figure shows a box plot illustrating the changes in cDNA yield when different macromolecular crowding effectors are added to the reverse transcription system.

[0054] By comparing the changes in cDNA yield after adding different types and concentrations of macromolecular crowding effect substances to the reverse transcription system, 2% Ficoll PM400 showed the best results.

[0055] Example 3: Preparation process: 1. Thawing frozen PBMCs (human peripheral blood mononuclear cells): Remove the frozen cells from liquid nitrogen and quickly transfer them to a 37°C water bath, stirring constantly until they are uniformly thawed. When the cells are thawed to the size of a mung bean, transfer the cell suspension to 9 volumes of DMEM medium containing 0.04% BSA. Gently pipette to mix, centrifuge at 350g for 5 minutes, wash once with DMEM medium containing 0.04% BSA, pass through a 40μm cell sieve, centrifuge at 350g for 5 minutes, remove dead cells using a Dead Cell Removal Kit, centrifuge at 350g for 5 minutes, resuspend the cells in PBS solution containing 0.04%–1% BSA and 0.1%–1% Pluronic F127, and use a cell counter to detect cell viability and concentration. Dilute the cells to 4.4 × 10⁶ cells / ml with PBS solution containing 0.04%–1% BSA and 0.1%–1% Pluronic F127, resulting in a cell viability of 88%.

[0056] Thaw the gel microspheres, DTT, and PK enzyme on an ice box, gently tap to mix, centrifuge, and then place on an ice box for later use. Take 5 μL of the diluted cell suspension (totaling 22,000 cells), add 5 μL of 20 U / μL SUPERase InRNase Inhibitor, gently pipette to mix, and place on an ice box for later use. Prepare the cell lysis mix by placing the pre-chilled sample tubes at -20°C on an ice box, pipette to mix, centrifuge, add 10 μL of the prepared cell suspension, quickly and gently pipette to mix, and add 200 μL of pre-chilled droplets at -20°C to generate oil.

[0057] Cell lysis Mix component table:

[0058] 2. Using a desktop shaker, set the prepared sample tube to the maximum speed of 3000 rpm and briefly oscillate horizontally for about 10 seconds. Then quickly switch to vertical oscillation for 1 minute. During vertical oscillation, gently press the side wall of the sample tube against the edge of the shaker rotor and oscillate by lightly touching the side wall of the sample tube with the edge of the shaker rotor. Do not pause during oscillation. After oscillation, let it stand for 1 minute to remove 90% of the bottom oil phase. When removing the oil phase, vent the pipette to the first setting and slowly insert the pipette tip along the inner wall of the tube into the upper layer of the oil phase. Vent the pipette to the second setting and expel the water-in-oil droplets from the tip opening. Only aspirate the oil phase portion. After removing the tip, gently press the area around the tip against the inner wall of the tube before discarding the oil phase.

[0059] 3. Transfer the generated water-in-oil emulsion sample tube to an incubation container for lysis. The lysis procedure is as follows: incubate at 50°C for 30 min to lyse the cells, and then incubate at 80°C for 10 min to inactivate proteinase K.

[0060] 4. Demulsification, washing, and purification of microspheres.

[0061] A. Prepare the demulsifying buffer solution and washing buffer solution according to the demulsifying buffer solution composition table and the washing buffer solution composition table.

[0062] Demulsifying buffer component table:

[0063] Washing buffer composition table:

[0064] B. Slowly add 180 μL of demulsification buffer and 40 μL of perfluorooctol (1H,1H,2H,2H-perfluoro-1-octanol, Sigma catalog number 370533) along the side wall of the sample tube. Invert the tube 10-20 times to mix until demulsification is complete (vortex for 5 seconds and then briefly centrifuge if necessary). If the solution is not homogeneous (demulsification is incomplete), add another 5-20 μL of perfluorooctol, repeat the inversion and mixing process, and briefly centrifuge using a desktop centrifuge (pause centrifugation by turning off the power).

[0065] C. After centrifugation, the solution will separate into three layers: a lower oil phase, a middle bead layer, and an upper aqueous phase. Thoroughly remove the bottom oil phase (if incomplete removal results in unbroken emulsion droplets that inhibit the reverse transcription reaction; if necessary, a small amount of microspheres can be aspirated to ensure complete oil phase removal).

[0066] D. Transfer all remaining solution to a 1.5 ml EP tube. Use 1 ml of washing buffer to wash the microspheres three times by resuspending, centrifuging, and discarding the supernatant. After washing, let stand for 1 minute. The gel microspheres will precipitate at the bottom of the tube. Remove the water layer, transfer the remaining microspheres and buffer to a 0.2 ml EP tube, centrifuge again, remove residual buffer to the 50 μL mark (marked on the EP tube beforehand), and store in an ice box for later use.

[0067] E. Release molecular tag sequence, reverse transcription, magnetic bead purification.

[0068] E1. Prepare the reverse transcription mix: Add 50 μL of reverse transcription mix to the microtube and gently pipette to mix.

[0069] Reverse Transcription Mix Components Table:

[0070] E2. Irradiate the sample tube containing the reverse transcription system with ultraviolet light to break the photosensitive chemical bonds of the microspheres, releasing the molecular barcode sequence on the microspheres and the mRNA it captures. Gently blow and mix well.

[0071] E3. Perform reverse transcription in a PCR instrument. Reverse transcription program: 105℃ with hot lid, 100ul volume, 25℃ / 5min, 42℃ / 90min, 85℃ / 10min, 4℃ hold.

[0072] E4. Transfer the supernatant to a new EP tube. Add 120 μl of washing buffer to the remaining bead tube, mix by pipetting, and then briefly centrifuge. Stop centrifugation by turning off the power and let it stand for 1 minute. Then, transfer the supernatant to a new EP tube.

[0073] E5. Purify the reverse transcription system by removing microspheres using 1.5~2× DNA magnetic beads, and reconstitute the product using 46μL EB.

[0074] F. cDNA amplification & purification.

[0075] F1. Prepare the cDNA amplification mix. Add 54 μL of cDNA amplification mix to the purified product.

[0076] Reverse Transcription Mix Components Table:

[0077] F2. Perform cDNA amplification in a PCR instrument. cDNA amplification program: 105℃ hot cap, 100μl volume, 95℃ / 3min, 98℃ / 15s, 67℃ / 20s, 72℃ / 3min for 20 cycles, 72℃ / 5min, 4℃ hold.

[0078] F3. Purify the amplification product using 0.6× DNA magnetic beads, repeat the purification process once, and reconstitute the cDNA using 40 μL EB.

[0079] G. Library construction and sequencing: Use the Nextera XT DNA library preparation kit or other alternative library construction systems, along with primers that match the microsphere molecular tag sequence, to construct the library.

[0080] H. Sequencing and Analysis: After successful library construction, sequencing was performed on the Illumina sequencing platform. Following sequencing, the data was analyzed using appropriate software.

[0081] I. Experimental Results.

[0082] I1, see appendix Figure 4 Microscopic examination revealed that, in the water-in-oil emulsion generated by oscillation, the probability of a single droplet containing multiple colloidal beads was less than 1%. With a sample volume of approximately 500,000 microbeads and 22,000 cells, the percentage of multiple cells in the generated effective droplets (containing both cells and microbeads) was less than 1%. Figure 4Images of water-in-oil droplets generated by oscillation using PIP technology are shown.

[0083] I2, see appendix Figure 5 The amplified and purified cDNA product was analyzed using an Agilent 2100 bioanalyzer. The main peak fragments were distributed between 0.8 and 1.5 kb. (See attached figure.) Figure 5 The diagram shows a comparison between the peak diagrams of cDNA synthesized using microfluidic single-cell technology and the peak diagrams of cDNA synthesized using the PIP single-cell technology of this invention. The comparison shows that the cDNA products synthesized by the two single-cell technologies have a similarity of more than 80%.

[0084] I3. The quantitative parameters of the PIP single-cell library and the microfluidic single-cell library constructed from the same sample are compared as follows:

[0085] Comparative analysis of the single-cell library quantitative parameters of Example 3 and the microfluidic platform control showed that, with the same amount of data and similar number of cells obtained, the total number of genes obtained by PIP single-cell library was 95% of that of microfluidic single-cell library, the cell capture rate was 92% of that of microfluidic single-cell library, the median number of genes in cells was 72.8% of that of microfluidic single-cell library, and the median number of UMIs in cells was 66% of that of microfluidic single-cell library. There were certain differences between the two.

[0086] Figure 6 The diagram shows the violin plots of the number of gene types in cells of a microfluidic single-cell library constructed from the same sample versus the PIP single-cell library of this invention. By comparing the distribution of the number of gene types in cells of the two libraries, the distribution trends of gene types in cells obtained by the two techniques are basically the same, but the transcript richness obtained by the PIP technique is lower than that of the microfluidic single-cell library.

[0087] Figure 7 The differences in cell clusters identified by the microfluidic single-cell library constructed from the same sample versus the PIP single-cell library of this invention are shown. Comparative analysis revealed that the two libraries identified the same cell types, with each cell type exhibiting a similarity of over 80%.

[0088] Based on the above analysis, although the PIP single-cell sequencing technology of this invention differs somewhat from the microfluidic single-cell technology, with the obtained transcript abundance being about 70%, its cell capture rate, distribution of intracellular gene types, and identification of cell types and their proportions are more than 80% similar to the microfluidic platform.

[0089] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.

Claims

1. A non-instrument-dependent high-throughput single-cell library construction method, characterized in that, Includes the following steps: S1: Water-in-oil droplet generation: Based on the principle of sequential partitioning of particle template emulsification, in a low-temperature environment, cell suspension, light-triggered microspheres carrying molecular tag sequences, and temperature-controlled cell lysis reagent are mixed and then oil is added to generate droplets. Water-in-oil droplets are generated through oscillation. S2: Temperature-controlled cell lysis: The water-in-oil system is heated to activate the lysis reagent and induce cell lysis. S3: Demulsification and microsphere washing: Demulsification buffer and perfluorooctyl alcohol are added to break the emulsion, and washing is performed to remove ineffective mRNA; S4: Molecular tag release and reverse transcription: Microspheres are stimulated by specific light intensity to release molecular tag sequences and their captured mRNA, which are then used for cDNA synthesis in an optimized reverse transcription system; S5: cDNA amplification and purification: cDNA is amplified using a PCR amplification system, and the product is purified using magnetic beads; S6: Library construction and sequencing: Construct single-cell libraries using a library construction kit and perform sequencing.

2. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, In S1, the cell suspension is resuspended in DPBS solution containing 0.04%~0.1% BSA and 0.1%~1% Pluronic F127; while the temperature-controlled cell lysis reagent has a final concentration of 30~300 U / ml proteinase K, 20~70 mMDTT, 0.01%~0.1% BSA and 1%~10% Plusion® HF Buffer.

3. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, In S1, the number of light-triggered microspheres is more than 10 times the number of cells; the volume of droplet-generated oil is more than 1.5 times the volume of the aqueous phase; the oscillation operation uses a desktop shaker at a maximum speed of 3000 rpm, first oscillating horizontally for 10 seconds, then oscillating vertically for 1-2 minutes, with the edge of the shaker rotor lightly touching the side wall of the centrifuge tube.

4. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, The phototriggered microspheres in S1 are magnetic beads or gel microspheres carrying molecular tag sequences. These molecular tag sequences are linked by photosensitive chemical bonds and include R1 sequencing primers, cell barcodes, UMI, and mRNA capture sequences.

5. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, The temperature-controlled lysis procedure for S2 is as follows: incubate at 37℃~60℃ for 10~30 minutes to lyse the cells, and then incubate at 80℃~85℃ for 5~10 minutes to inactivate proteinase K; all operations before S2 lysis are performed in a low-temperature environment of ≤18℃.

6. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, The demulsification buffer in S3 consists of 250 mM Tris-HCl, 375 mM KCl, 15 mM MgCl2, and 50 mM DTT; perfluorooctyl alcohol is added for demulsification, and the mixture is inverted and mixed 10-20 times.

7. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, The washing buffer in S3 consists of 100 mM Tris-HCl, 150 mM KCl, 6 mM MgCl2, 20 mM DTT, and 1% Poloxamer™ 188. The microsphere washing process involves resuspension, centrifugation, and discarding the supernatant 3-4 times. All centrifugation steps are performed by turning off the power and pausing the centrifugation. After a short centrifugation, the microspheres are allowed to stand and separate into layers.

8. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, The reverse transcription system in S4 contains: 2%~4% Ficoll PM400, 1~5 μM MTO, 0.5~1 mM dNTPs, 0.5~2 U / μL LRNase inhibitor, 1~10 U / μL Maxima HMinus reverse transcriptase, and 1×RT Buffer; the reverse transcription program in S4 is: 25℃ / 5 min, 42℃ / 90 min, and 85℃ / 10 min; the molecular tag release in S4 is achieved by breaking the photosensitive chemical bonds through specific light intensity irradiation, releasing the molecular tag sequence on the microspheres and the mRNA it captures.

9. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, In S5, cDNA amplification was performed using the KAPAHiFiHotStartReadyMixPCRKit. The amplification program was as follows: First, initial denaturation was performed at 95°C for 3 minutes. The process was then repeated 20 times. Each cycle consisted of the following steps: denaturing the DNA at 98°C for 15 seconds, annealing the primers at 67°C for 20 seconds, extending the DNA strand at 72°C for 3 minutes, and finally extending the DNA strand at 72°C for 5 minutes. In S5, the DNA was purified twice using 0.6-0.8× DNA magnetic beads.

10. The non-instrument-dependent high-throughput single-cell library construction method according to claim 1, characterized in that, The S6 Chinese library was constructed using the NexteraXT DNA library construction kit, along with primers that matched the microsphere molecular tags.

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