Preparation method of nucleic acid coded beads for single bacterium / single cell sequencing
By using gelatin and epoxy compounds to modify the preparation of nucleic acid-encoded beads, the shortcomings of existing materials in single-bacterial/single-cell sequencing are overcome, resulting in more efficient sequencing data quality and sensitivity.
Patent Information
- Application Number
- CN202511242013.4
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-09-02
- Publication Date
- 2025-11-21
AI Technical Summary
Existing coding bead materials suffer from problems such as insufficient mechanical strength, low chemical modification efficiency, and non-specific adsorption in single-bacterial/single-cell sequencing, which affect the purity and sensitivity of sequencing data.
Using gelatin as the main material for coding beads, nucleic acid-coded beads with larger pores and higher chemical stability were prepared through alkalization treatment and epoxy compound modification. The size and surface modification of the gel beads were controlled using a microfluidic system.
This invention achieves green, non-toxic, and structurally stable coding beads, simplifies the preparation process, avoids the introduction of impurities, improves the stability of nucleic acids and enzymes, and enhances the permeability of the reaction system.
Smart Images
Figure CN120989216A_ABST
Abstract
Description
TECHNICAL FIELD
[0001] The present application relates to the technical field of biological materials, in particular to a preparation method of nucleic acidized coding beads for single bacterial / single cell sequencing. BACKGROUND
[0002] Single bacterial / single cell sequencing is a powerful technology for discovering heterogeneous cell subpopulations and identifying cell states, which uses similar barcoding strategies to uniquely label the entire genome or transcriptome of each cell in the population. In the past 10 years, droplet-based microfluidic technology has been widely used for the separation of single bacteria / single cells and the sequencing of nucleic acids, which mainly includes inDrop and Drop-seq methods. In inDrop, a single cell, a reverse transcription / cleavage mixture and a coding bead are co-encapsulated into a droplet reactor, and after cell lysis, the barcoding sequences on both sides of the reverse transcription primer are released from the coding bead to initiate the reverse transcription process. The Drop-seq method is to hybridize the transcriptome from a single cell to the coding bead after cell lysis. The preparation of the coding bead is particularly critical, which gives each single bacterial / single cell or single cell nucleus a unique molecular tag, so as to accurately distinguish the genetic information of different cells after sequencing. The main coding bead materials at present mainly include polyacrylamide, silica and polystyrene, although these materials have been widely used in single bacterial / single cell sequencing, but their inherent defects limit the further improvement of the technology. For example: 1. Polyacrylamide has controllable mechanical strength, but due to the dense polymer network generated by the end crosslinking of low molecular weight monomers, the surface pore size of the synthesized coding bead is only a few nanometers, which is not conducive to the entry of polymerase and the coupling of some nucleic acid fragments. In addition, its dissolution process (such as DTT reduction) may introduce additional impurities, affecting the purity of the sequencing data. 2. The surface chemical modification efficiency of silica magnetic beads is high, but the rigid structure is easy to cause cell mechanical stress damage, and the high density characteristics limit the microfluidic sorting efficiency. 3. Polystyrene microspheres have excellent chemical stability, but the hydrophobic surface is easy to cause non-specific adsorption, which leads to an increase in background noise, especially in the detection of low-abundance targets, the sensitivity is significantly reduced.
[0003] Gelatin, as an animal-derived protein, is considered as an ideal candidate material for coding beads due to its excellent biocompatibility, low-temperature gelation characteristics and controllable pore structure. However, traditional gelatin microspheres mainly fix nucleic acid probes through physical embedding or simple adsorption, which leads to low labeling efficiency, easy probe shedding and difficulty in realizing high-density coding. SUMMARY
[0004] The purpose of the present application is to provide a preparation method of nucleic acidized coding beads for single bacterial / single cell sequencing to solve the problems raised in the background.
[0005] To achieve the above object, the present application provides the following technical scheme: a preparation method of nucleic acid coded beads for single bacterium / single cell sequencing, comprising:
[0006] S1: dissolving gelatin in deionized water at 40-50 DEG C to prepare a 0.1%-10% gelatin solution;
[0007] S2: cooling the gelatin solution in S1 to 30-35 DEG C, adding sodium hydroxide standard titration solution to the gelatin solution for alkalization treatment, and adjusting the pH of the gelatin solution to 10-13;
[0008] S3: stirring the gelatin solution after alkalization treatment, slowly adding a compound containing an epoxy feature to the gelatin solution at a molar ratio of 1:1-10:1 within 10-30 min, and maintaining the temperature at 30-35 DEG C for 12-15 h of stirring reaction;
[0009] S4: continuing to maintain the temperature of the gelatin solution in S3 at 30-35 DEG C, washing with an organic solvent for multiple times to remove excess compound containing an epoxy feature, and collecting the gelatin solution for freeze-drying for 24-48 h;
[0010] S5: dissolving the dried gelatin powder in deionized water at 40-50 DEG C to prepare a 0.1%-10% gelatin solution;
[0011] S6: continuing to cool the completely dissolved gelatin solution to 30-35 DEG C, and:
[0012] firstly adding a nucleic acid modified with a specific group for 1-2 h to obtain a mixed solution, and preparing the mixed solution into gel beads with a diameter of 20-200 μm through a microfluidic system; or firstly preparing the gelatin solution into gel beads with a diameter of 20-200 μm through a microfluidic system, and then adding a nucleic acid modified with a specific group for 1-2 h.
[0013] Preferably, in S2, the alkalization treatment time is 0.5-1 h.
[0014] Preferably, in S3, the compound containing an epoxy feature is 1,2-epoxyheptane, 1,2-epoxybutane or (S)-2-(chloromethyl)oxirane.
[0015] Preferably, in S3, the compound containing an epoxy feature needs to be dissolved in dimethyl sulfoxide first, and the volume ratio of the compound containing an epoxy feature to dimethyl sulfoxide is 1:2-1:99.
[0016] Preferably, in S4, the organic solvent is anhydrous ethanol, DMF or dimethyl sulfoxide.
[0017] Preferably, in S6, the nucleic acid modified with a specific group is an amino-modified nucleic acid.
[0018] Compared with the prior art, the present application has the following advantages:
[0019] 1. Using gelatin as the main material of the coding beads, which is green, non-toxic and stable in structure, and will not destroy nucleic acids and enzymes in the reaction.
[0020] 2. The beading method is simple, and the gel beads are only controlled by physical methods, without adding other compounds, so as not to introduce unnecessary impurities into the sequencing system.
[0021] 3. The coding beads made of gelatin have larger surface pore size, which is more conducive to the entry and exit of components in the reaction system. BRIEF DESCRIPTION OF DRAWINGS
[0022] Figure 1 Flow chart for nucleic acid gelatin synthesis;
[0023] Figure 2 Infrared spectrum of gelatin and gelatin-epoxy butane, wherein (1) is gelatin; (2) is gelatin-epoxy butane.
[0024] Figure 3 NMR spectrum of gelatin and gelatin-epoxy butane, wherein (1) is gelatin; (2) is gelatin-epoxy butane.
[0025] Figure 4 Fluorescence picture of FITC-dextran polymer with a molecular weight of 70 kDa entering the gelatin gel beads, scale 100 μm.
[0026] Figure 5 Fluorescence picture of FITC-dextran polymer with a molecular weight of 150 kDa entering the gelatin gel beads, scale 100 μm.
[0027] Figure 6 Fluorescence picture of FITC-dextran polymer with a molecular weight of 400 kDa entering the gelatin gel beads, scale 100 μm.
[0028] Figure 7 Gel electrophoresis result, a is mark 100 bp, b and c are specific sequences. DETAILED DESCRIPTION
[0029] The technical solutions in the embodiments of the present application will be described clearly and completely below with reference to the drawings in the embodiments of the present application. Obviously, the described embodiments are only part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.
[0030] Please refer toFigures 1-7 A method for preparing nucleic acid coded beads for single bacterial / single cell sequencing, comprising:
[0031] S1: Dissolve gelatin in deionized water at 40-50°C to prepare a 0.1%-10% gelatin solution.
[0032] S2: Cool the gelatin solution in S1 to 30-35°C, and add a standard sodium hydroxide titration solution to perform alkalization treatment, and adjust the pH of the gelatin solution to 10-13; the alkalization treatment time is 0.5-1 h.
[0033] S3: Stir the gelatin solution after alkalization treatment, and slowly add a compound containing an epoxy feature to the gelatin solution at a molar ratio of 10:1 within 10-30 min, and maintain the temperature at 30-35°C for 12-15 h of stirring reaction.
[0034] The compound containing an epoxy feature is 1,2-epoxyheptane, 1,2-epoxybutane, or (S)-2-(chloromethyl)oxirane.
[0035] The compound containing an epoxy feature needs to be dissolved in anhydrous ethanol first, and the volume ratio of the compound containing an epoxy feature to anhydrous ethanol is 1:2-1:99.
[0036] S4: Continue to maintain the temperature of the gelatin solution in S3 at 30-35°C, and use an organic solvent to wash multiple times to remove excess compound containing an epoxy feature, and collect the gelatin solution for freeze-drying for 24-48 h.
[0037] The organic solvent is anhydrous ethanol, DMF, or dimethyl sulfoxide.
[0038] S5: Redissolve the dried gelatin powder in deionized water at 40-50°C, and prepare a 0.1%-10% gelatin solution.
[0039] S6: Continue to cool the completely dissolved gelatin solution to 30-35°C, add a nucleic acid modified with a specific group for 1-2 h, and prepare a 20-200 μm gel bead through a microfluidic system.
[0040] The nucleic acid modified with a specific group is an amino-modified nucleic acid.
[0041] In S6, in addition to the above sequence, the gelatin solution can also be prepared into a 20-200 μm gel bead first, and then a nucleic acid modified with a specific group is added for 1-2 h.
[0042] Unless otherwise specified, all reagents in this experiment are purchased from Aladdin.
[0043] Example 1
[0044] (1) 0.01 mmol (0.1 g) gelatin was dissolved in 100 ml deionized water, heated to 40 °C with stirring until the gelatin was completely dissolved and the solution became transparent from turbid;
[0045] (2) The gelatin solution was cooled to 30 °C, and 3.500 mol / L sodium hydroxide standard titration solution was added to adjust the pH of the gelatin solution to 10, and the alkalization reaction was carried out for 0.5 h;
[0046] (3) The solution of step (2) was continuously stirred, and 0.1 mmol (0.0072 g) 1,2-epoxybutane was dissolved in 0.713 ml anhydrous ethanol and slowly added dropwise within 10 min, the solution temperature was maintained at 30 °C and the reaction was continued for 12 h;
[0047] (4) The solution obtained in step (3) was continuously maintained at 30 °C, and the gelatin solution was washed with 90 ml anhydrous ethanol in 3 times, and the gelatin solution was collected and freeze-dried for 24 h to obtain gelatin-epoxybutane powder.
[0048] (5) 0.0001 mmol (0.001 g) gelatin-epoxybutane powder was taken and added to 1 ml deionized water, heated to 40 °C until the powder was completely dissolved to obtain a gelatin-epoxybutane solution with a concentration of 0.1%.
[0049] (6) The 0.1% gelatin-epoxybutane solution in step (5) was cooled to 30 °C, and 0.001 mmol amino-modified nucleic acid was added, and the reaction was carried out for 1 h to obtain a 0.1% gelatin-epoxybutane-nucleic acid solution.
[0050] (7) The solution after the reaction of step (6) was prepared into gel beads with a diameter of 20 μm using a microfluidic system, and the specific parameters of the microfluidic system were as follows: 1. The aqueous phase was a 0.1% gelatin-epoxybutane-nucleic acid solution, and the flow rate was 50 μl / h; 2. The oil phase was a fluorine oil (Novec 7500 Engineered Fluid, 3M) containing 1% surfactant, and the flow rate was 100 μl / h; 3. The chip channel width was 20 μm. The collected gel beads were kept at 4 °C for 1 h, and then washed with fluorine oil containing 20% 1H, 1H, 2H, 2H-perfluoro-1-octanol to obtain nucleic acid-modified gelatin gel beads and then dispersed in 1 ml deionized water.
[0051] The process of coupling gelatin with amino-modified nucleic acid is shown in Figure 1 After the gelatin was grafted with 1,2-epoxybutane as shown in Figure 2 , the characteristic peak at 1651 cm -1 was split into 1656 cm -1 and 1600 cm -1two peaks, which is due to 1,2-epoxybutane directly linked to the pyran ring on the gelatin, resulting in the splitting of the C-O stretching vibration peak. As shown in the nuclear magnetic resonance Figure 3 The substitution rate was calculated to be 9.8% according to the integral area of the end group proton peak at 2.47 ppm and 5-5.2 ppm of the gelatin grafted with 1,2-epoxybutane, as shown in the nuclear magnetic resonance
[0052] To test the pore size of the gel beads, 50 ul of the above gel bead aqueous solution was placed in 1 ml of FITC-dextran solution with a concentration of 2 mg / ml and stirred for 2 h, where the molecular weight of FITC-dextran was three, 70 kDa, 150 kDa and 400 kDa. After stirring, the gel beads were centrifuged at 2000 rpm for 5 min and washed 3 times, and a fluorescence microscope was used to take a fluorescence picture. As shown in Figures 3-5 It is shown that the FITC-dextran polymers with molecular weights of 70 kDa and 150 kDa can freely enter the nucleic acidized gelatin gel beads.
[0053] Example 2
[0054] (1) 0.5 mmol (5 g) of gelatin was dissolved in 50 ml of deionized water, and stirred and heated to 50°C until the gelatin was completely dissolved, and the solution changed from turbid to transparent;
[0055] (2) The above gelatin solution was cooled to 35°C, and a standard sodium hydroxide titration solution with a concentration of 3.500 mol / L was added to adjust the pH of the gelatin solution to 13, and the alkalization reaction was carried out for 1 h;
[0056] (3) The solution of step (2) was continuously stirred, 0.5 mmol (0.036 g) of 1,2-epoxybutane was dissolved in 0.072 ml of anhydrous ethanol and slowly added within 30 min, the solution temperature was maintained at 35°C and the reaction was continued for 15 h;
[0057] (4) The solution obtained in step (3) was continuously maintained at a temperature of 35°C, and the gelatin solution was washed with 45 ml of DMF in 3 times, and the gelatin solution was collected and freeze-dried for 48 h to obtain a gelatin-epoxybutane powder.
[0058] (5) 0.01 mmol (0.1 g) of gelatin-epoxybutane powder was taken and added to 1 ml of deionized water, and heated to 50°C until the powder was completely dissolved to obtain a gelatin-epoxybutane solution with a concentration of 10%.
[0059] (6) 1 ml of 10% gelatin- butylene oxide solution was prepared into gel beads with a diameter of 200 μm using a microfluidic system. The specific parameters of the microfluidic system were as follows: 1. The water phase was a 10% gelatin-butylene oxide-nucleic acid solution, and the flow rate was 50 μl / h; 2. The oil phase was a fluorine oil (Novec 7500 Engineered Fluid, 3M) containing 1% surfactant, and the flow rate was 100 μl / h; 3. The chip channel width was 200 μm. The collected gel beads were kept at 4°C for 1 h, washed with fluorine oil containing 20% 1H, 1H, 2H, 2H-perfluoro-1-octanol, and dispersed in 5 ml of nuclease-free water.
[0060] (7) 0.1 mmol of amino-modified nucleic acid was added to 5 ml of gel bead aqueous solution at room temperature, and the reaction was carried out for 2 h. The unreacted nucleic acid was removed by centrifugation at 2000 rpm for 5 min, and nucleic acid-modified gelatin gel beads were obtained and redispersed in 1 ml of nuclease-free water.
[0061] Example 3
[0062] (1) 1 mmol (10.0 g) of gelatin was dissolved in 200 ml of deionized water, and the solution was heated to 45°C with stirring until the gelatin was completely dissolved, and the solution changed from turbid to transparent;
[0063] (2) The gelatin solution was cooled to 33°C, and a standard titration solution of sodium hydroxide with a concentration of 3.50 mol / L was added to adjust the pH of the gelatin solution to 12, and the alkalization reaction was carried out for 45 min;
[0064] (3) The solution of step (2) was continuously stirred, and 5 mmol (0.36 g) of 1,2-butylene oxide was dissolved in 18 ml of anhydrous ethanol and slowly added dropwise within 20 min, and the solution temperature was maintained at 33°C and the reaction was continued for 13 h;
[0065] (4) The solution obtained in step (3) was continuously maintained at a temperature of 33°C, and the gelatin solution was washed with 180 ml of dimethyl sulfoxide in three portions, and the gelatin solution was collected and freeze-dried for 36 h to obtain gelatin-butylene oxide powder.
[0066] (5) 0.01 mmol (0.10 g) of gelatin-butylene oxide powder was added to 2 ml of nuclease-free water, and the temperature was raised to 45°C until the powder was completely dissolved to obtain a 5% gelatin-butylene oxide solution.
[0067] (6) 1 ml of 5% gelatin-butane solution was used to prepare gel beads with a diameter of 100 μm using a microfluidic system. The specific parameters of the microfluidic system were: 1. Aqueous phase: 5% gelatin-butane-nucleic acid solution, flow rate: 50 μl / h; 2. Oil phase: fluorinated oil (Novec 7500 Engineered Fluid, 3M) containing 1% surfactant, flow rate: 100 μl / h; 3. Chip channel width: 100 μm. The collected gel beads were kept at 4℃ for 1 h, washed with fluorinated oil containing 20% 1H,1H,2H,2H-perfluoro-1-octanol, and dispersed in 5 ml of nuclease-free water.
[0068] (7) Add 0.1 mmol of amino-modified nucleic acid to 5 ml of gel bead aqueous solution at room temperature, react for 1.5 h, centrifuge at 2000 rpm for 5 min to remove unreacted nucleic acid, and obtain nucleic acid-modified gelatin gel beads, which are then redispersed in 1 ml of nuclease-free water.
[0069] Example 4
[0070] (1) Dissolve 0.6 mmol (6.0 g) of gelatin in 100 ml of deionized water, stir and heat to 43 °C until the gelatin is completely dissolved, and the solution changes from turbid to transparent;
[0071] (2) Cool the above gelatin solution to 32°C, add 3.50 mol / L sodium hydroxide standard titration solution, adjust the pH of the gelatin solution to 11, and carry out the alkalization reaction for 40 min;
[0072] (3) Continue stirring the solution from step (2), dissolve 3.6 mmol (0.411 g) of 1,2-epoxyheptane in 24 ml of anhydrous ethanol and slowly add it dropwise over 25 min, keep the solution temperature at 32 °C and continue the reaction for 14 h;
[0073] (4) The solution obtained in step (3) is kept at 32°C, and the gelatin solution is washed three times with 90 ml of anhydrous ethanol. The gelatin solution is collected and freeze-dried for 35 h to obtain gelatin-epoxyheptane powder.
[0074] (5) Take 0.006 mmol (0.060 g) of gelatin-epoxyheptane powder, add 1 ml of enzyme-free water, heat to 48 °C until the powder is completely dissolved, and obtain a gelatin-epoxyheptane solution with a concentration of 6%.
[0075] (6) 1 ml of 6% gelatin-epoxy heptane solution was prepared into gel beads with a diameter of 50 μm using a microfluidic system. The specific parameters of the microfluidic system were as follows: 1. The water phase was a 6% gelatin-epoxy heptane-nucleic acid solution, and the flow rate was 50 μl / h; 2. The oil phase was a fluorine oil (Novec 7500 Engineered Fluid, 3M) containing 1% surfactant, and the flow rate was 100 μl / h; 3. The chip channel width was 50 μm. The collected gel beads were kept at 4°C for 1 h, washed with a fluorine oil containing 20% 1H, 1H, 2H, 2H-perfluoro-1-octanol, and dispersed in 5 ml of nuclease-free water.
[0076] (7) 0.1 mmol of amino-modified nucleic acid was added to 5 ml of gel bead aqueous solution at room temperature, and the reaction was carried out for 100 min. The unreacted nucleic acid was removed by centrifugation at 2000 rpm for 5 min, and the nucleic acid-modified gelatin gel beads were obtained and redispersed in 1 ml of nuclease-free water.
[0077] Example 5
[0078] (1) 0.3 mmol (3.0 g) of gelatin was dissolved in 100 ml of deionized water, and the solution was heated to 48°C while stirring until the gelatin was completely dissolved, and the solution changed from turbid to transparent;
[0079] (2) The gelatin solution was cooled to 34°C, and 3.50 mol / L sodium hydroxide standard titration solution was added to adjust the pH of the gelatin solution to 11, and the alkalization reaction was carried out for 50 min;
[0080] (3) The solution of step (2) was continuously stirred, and 0.9 mmol (0.083 g) of (S)-2-(chloromethyl)oxirane was dissolved in 2.4 ml of anhydrous ethanol and slowly added dropwise within 15 min, and the solution temperature was maintained at 34°C and the reaction was continued for 14 h;
[0081] (4) The solution obtained in step (3) was continuously maintained at a temperature of 34°C, and the gelatin solution was washed with 90 ml of anhydrous ethanol in three portions, and the gelatin solution was collected and freeze-dried for 40 h to obtain gelatin-(chloromethyl)oxirane powder.
[0082] (5) 0.003 mmol (0.030 g) of gelatin-(chloromethyl)oxirane powder was added to 1 ml of nuclease-free water, and the temperature was raised to 48°C until the powder was completely dissolved, to obtain a gelatin-(chloromethyl)oxirane solution with a concentration of 3%.
[0083] (6) 1 ml of 3% gelatin-(chloromethyl)oxirane solution was prepared into gel beads with a diameter of 150 pm using a microfluidic system. The specific parameters of the microfluidic system were as follows: 1. The aqueous phase was a 3% gelatin-(chloromethyl)oxirane-nucleic acid solution, and the flow rate was 50 pl / h; 2. The oil phase was a fluorine oil (Novec 7500 Engineered Fluid, 3M) containing 1% surfactant, and the flow rate was 100 pl / h; 3. The chip channel width was 150 pm. The collected gel beads were kept at 4°C for 1 h, washed with fluorine oil containing 20% 1H, 1H, 2H, 2H-perfluoro-1-octanol, and dispersed in 5 ml of nuclease-free water.
[0084] (7) 0.1 mmol of amino-modified nucleic acid was added to 5 ml of gel bead aqueous solution at room temperature, and the reaction was carried out for 100 min. The unreacted nucleic acid was removed by centrifugation at 2000 rpm for 5 min, and the nucleic acid-modified gelatin gel beads were obtained and redispersed in 1 ml of nuclease-free water.
[0085] Example 6
[0086] This example is the application of nucleic acid-modified gelatin gel beads in single bacterial / single cell sequencing
[0087] (1) The nucleic acid-modified gelatin gel beads in Example 1 (in which the amino-modified nucleic acid used was NH2-primer, ordered from Kingsrosa Biotech Co., Ltd.; amino-modified-5' TAAGCATTUGTAAAAAGATTAUUTTATAGGATATTAGA 3') were filtered using a 40 pm bacterial / cell filter. Then a pre-cooled washing buffer was used to facilitate the passage of the beads through the filter. The gel beads that passed through the filter were centrifuged at 2000 rpm for 5 min and 1 ml of washing buffer was added to the centrifuge tube (washing buffer: mix 980 ml of nuclease-free water, 10 ml of 1M Tris-HCl (pH 8.0), 200 pl of 0.5M EDTA and 10 ml of 10% Tween-20), and washed 3 times.
[0088] (2) 2.9 ml of 10x isothermal amplification buffer (NEB, B0537S), 2.3 ml of nuclease-free water and 0.8 ml of dNTP (10 mM each) were added to the centrifuge tube and vortexed thoroughly.
[0089] (3) The above solution and gel beads were equally divided into a round-bottom 96-well plate, 30 pl per well. The specific sequence primer (50 mM) was thawed at room temperature, and the specific sequence was added to the 96-well plate, 15 pl per well, and the ligation reagent was added to the 96-well plate, 20 pl per well, which included DNA ligase, isothermal amplification buffer, dNTP.
[0090] (4) Place the 96-well plate in the PCR machine and incubate at 37°C for 120 minutes.
[0091] (5) After incubation, add stop buffer to the 96-well plate, 20 μL per well. Collect all solutions and gel beads from all wells and incubate at room temperature for 30 minutes. (Stop buffer: 880 ml of nuclease-free water, 10 ml of 1 M Tris-HCl (pH 8.0), 50 ml of 0.5 M EDTA, 10 ml of 10% (vol / vol) Tween-20, and 50 ml of 2 M KCl)
[0092] (6) After the reaction is stopped, centrifuge and discard the supernatant, and wash the beads three times with wash buffer.
[0093] The process quality of the nucleic acidized agar gel beads was detected. After the gel beads were quickly warmed to 50°C to melt the gel beads, the USER enzyme was incubated at 37°C for 20 minutes, the nucleic acidization was released, and analysis was performed by 2% gel electrophoresis. The results are shown in Figure 6
[0094] The above shows and describes the basic principles and main features of the present application and the advantages of the present application. It is obvious to those skilled in the art that the present application is not limited to the details of the above exemplary embodiments, and the present application can be implemented in other specific forms without departing from the spirit or essential characteristics of the present application. Therefore, the embodiments should be considered as exemplary and non-limiting, and the scope of the present application is defined by the appended claims rather than the above description, and therefore all changes falling within the meaning and scope of the equivalent elements of the claims are intended to be included in the present application, and any reference signs in the claims should not be considered as limiting the claims.
[0095] Although embodiments of the present application 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 therein without departing from the principles and spirit of the present application, and the scope of the present application is defined by the appended claims and their equivalents.
Claims
1. A method for preparing nucleic acid coded beads for single bacterium / single cell sequencing, characterized in that, The application relates to a method for preparing a gelatin gel bead, which comprises the following steps: S1: dissolving gelatin in deionized water at 40-50 DEG C to prepare a 0.1%-10% gelatin solution; S2: cooling the gelatin solution in S1 to 30-35 DEG C, adding a standard sodium hydroxide titration solution to the gelatin solution to perform alkalization treatment, and adjusting the pH of the gelatin solution to 10-13; S3: stirring the gelatin solution after the alkalization treatment, slowly adding a compound containing an epoxy feature in the gelatin solution at a molar ratio of 1:1-10:1 within 10-30 min, and maintaining the temperature at 30-35 DEG C to stir for 12-15 h; S4: continuing to maintain the temperature of the gelatin solution in S3 at 30-35 DEG C, washing the gelatin solution with an organic solvent for multiple times to remove the excess compound containing the epoxy feature, and collecting the gelatin solution to be freeze-dried for 24-48 h; S5: dissolving the dried gelatin powder in deionized water at 40-50 DEG C to prepare a 0.1%-10% gelatin solution; S6: cooling the completely dissolved gelatin solution to 30-35 DEG C, adding a nucleic acid modified with a specific group to the gelatin solution to react for 1-2 h to obtain a mixed solution, and preparing the mixed solution into a gel bead with a diameter of 20-200 mu m through a microfluidic system; or first preparing the gelatin solution into a gel bead with a diameter of 20-200 mu m through a microfluidic system, and then adding the nucleic acid modified with the specific group to the gelatin solution to react for 1-2 h. In S2, the alkalization treatment time is 0.5-1 h.
2. A method of preparing a nucleic acid coded bead for single bacterium / single cell sequencing as claimed in claim 1, wherein: In S3, the compound containing the epoxy feature is 1,2-epoxyheptane, 1,2-epoxybutane or (S)-2-(chloromethyl)oxirane.
3. A method of preparing a nucleic acid coded bead for single bacterium / single cell sequencing as claimed in claim 1, wherein: In S3, the compound containing the epoxy feature needs to be dissolved in anhydrous ethanol, and the volume ratio of the compound containing the epoxy feature to the anhydrous ethanol is 1:2-1:
99.
4. A method of preparing a nucleic acid coded bead for single bacterium / single cell sequencing as claimed in claim 1, wherein: In S4, the organic solvent is anhydrous ethanol, DMF or dimethyl sulfoxide.
5. A method of preparing a nucleic acid coded bead for single bacterium / single cell sequencing as claimed in claim 1, wherein: In S6, the nucleic acid modified with the specific group is an amino-modified nucleic acid.
6. A method of preparing a nucleic acid coded bead for single bacterium / single cell sequencing as claimed in claim 1, wherein: