A method for simultaneous construction of rabbit single cell immune repertoire TCR and BCR libraries based on droplet microfluidics

Abstract

The present invention relates to the field of biomedicine technology, and in particular to a method for simultaneously constructing a rabbit single-cell immune repertoire TCR and BCR library based on droplet microfluidics. The present invention designs a set of primers for the C region gene sequence of rabbit TCR and BCR / IG for the first time, and can complete the simultaneous construction of TCR and BCR libraries in the same reaction. It is currently the first solution for the construction of a rabbit single-cell immune repertoire library. Compared with existing single-cell immune repertoire solutions, the present invention is applicable to a wider range of cell input numbers, with cell input numbers as low as 200 and as high as 30,000. The present invention has a faster and more efficient library construction process.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a method for simultaneously constructing a rabbit single cell immune repertoire TCR and BCR library based on droplet microfluidics. Background Art

[0002] In the past decade, the development of single-cell technology has been very rapid. Single-cell transcriptome sequencing technology can help researchers obtain the gene expression patterns in individual cells of interest, and can also study the lineage relationship between these cells. Monoclonal antibodies are widely used in the field of biomedicine. Compared with traditional mouse antibodies, monoclonal and polyclonal antibodies derived from rabbits are increasingly favored by the pharmaceutical industry because of their many advantages. Compared with antibodies from other sources, rabbit antibodies have the following main advantages: 1. A wider antibody spectrum; 2. Simple immunoglobulin structure; 3. Higher specificity and affinity; 4. Candidate application of high-quality monoclonal antibody drugs. The spleen of rabbits contains 50 times more lymphocytes than the spleen of mice, so that the spleen of rabbits in a single experiment can obtain more antibodies with different antigen epitopes.

[0003] The rabbit immune system contains hundreds of millions of different T cells and B cells, and the diversity of TCRs and BCRs on them constitutes the TCR and BCR immune repertoires respectively. Each different TCR or BCR can recognize different antigens. The diversity of antigen recognition by TCR / BCR is generated during gene rearrangement, which is called V(D)J rearrangement. The core is that there are a large number of V, D and J gene fragments on the genome. The generation of TCR or BCR genes is the result of random combination of single V, D (optional) and J fragments. These different combinations form the basis of the highly variable variable region (V region). The β chain of TCR and the heavy chain of BCR are composed of VDJ fragments, while the α chain of TCR and the light chain of BCR are composed of VJ fragments. The hypervariable region (i.e., V region) is composed of 3 complementary determining regions (CDR) and 4 framework regions (FR), which are arranged alternately. Among them, the third complementary determining region (i.e., CDR3 region) has the highest diversity and best represents the specificity of the receptor library. The analysis of the paired αβ chain sequence of TCR in the rabbit T (B) cell clone that responds to a specific antigen (the sequence of paired heavy and light chains in BCR), especially the CDR3 sequence, is crucial for drug development and disease prevention represented by rabbit monoclonal antibodies. However, there has been no solution for how to simultaneously analyze the αβ chain (TCR) or heavy and light chain (BCR) in a single rabbit cell. At present, the methodology of the single-cell immune repertoire is mainly aimed at humans and mice. It is particularly important to design primer sequences that can target and capture the information of the rabbit single-cell immune repertoire for the rabbit genome. At present, there is no single-cell sequencing method suitable for simultaneously capturing the αβ chain in the rabbit TCR and the heavy and light chain in the BCR. Therefore, it is necessary to develop a method that can simultaneously capture the paired αβ chain in the TCR and the paired heavy and light chain in the BCR in a single rabbit cell. The present invention provides the first rapid and efficient method for simultaneously constructing a high-throughput rabbit single-cell TCR and BCR library. Summary of the invention

[0004] The purpose of the present invention is to solve the problem of the current blank method for constructing rabbit single-cell immune repertoires, and develop and design a set of primer sequences that can target the C region (constant region) gene sequences in rabbit TCR (T cell receptor) and BCR / IG (B cell receptor / antibody), combined with the applicant MobiNova-100 single-cell sequencing library construction system of Mozo Biotechnology (purchased from Mozo Biotechnology Co., Ltd., item number A1A40001; or reference patents CN202111667188.1, CN2 02111673080.3, CN202123452757.9, CN202123456944.4, CN202123452756.4) and MobiCube High-throughput Single-cell 5' Transcriptome Reagent Kit (Cat. No. S060100101, S060200101; or reference patent application No. 202311345771X), provide a method for quickly and efficiently constructing rabbit single-cell TCR and BCR libraries at the same time.

[0005] The present invention provides a method for simultaneously constructing a rabbit single cell immune repertoire TCR and BCR library based on droplet microfluidics, and the construction method comprises the following steps: S1. Synthesize primer sequences targeting rabbit TCR and BCR / IG, the primer sequences are shown in SEQ ID NO.5-40; S2. Preparation of single cell suspension; S3. Preparation and reverse transcription of single-cell oil-in-water droplets; S4. Demulsification and cDNA pre-amplification; S5. Rabbit TCR and BCR / IG related cDNA molecules were simultaneously enriched in the first round; S6. Rabbit TCR and BCR / IG related cDNA molecules were simultaneously enriched in the second round; S7. Construction of a mixed library of rabbit single-cell TCR and BCR based on Tn5 transposase.

[0006] In some embodiments, the primer sequences for the first round of enrichment are shown as SEQ ID NO.4 and SEQ ID NO.5-22, and the primer sequences for the second round of enrichment are shown as SEQ ID NO.4 and SEQ ID NO.23-40, wherein SEQ ID NO.4 is the forward primer in the PCR reaction, and SEQ ID NO.5-22 and SEQ ID NO.23-40 are both reverse primers in the PCR reaction.

[0007] In some embodiments, the first round of enrichment includes: taking the cDNA in S4 as a template, combining the TCR and BCR primers enriched in the first round as a mixture, and preparing a first round of enrichment PCR reaction solution, wherein the TCR primers in S5 include the Ampli-F primer sequence as shown in SEQ ID NO.4, the Ampli1-RaT-R1 primer sequence as shown in SEQ ID NO.5, the Ampli1-RaT-R2 primer sequence as shown in SEQ ID NO.6, the Ampli1-RaT-R3 primer sequence as shown in SEQ ID NO.7, and the Ampli1-RaT-R4 primer sequence as shown in SEQ ID NO.8, and the BCR primers in S5 include the Ampli1-RaB-R1 primer sequence as shown in SEQ ID NO.9, the Ampli1-RaB-R2 primer sequence as shown in SEQ ID NO.10, the Ampli1-RaB-R3 primer sequence as shown in SEQ ID NO.11, and the Ampli1-RaB-R4 primer sequence as shown in SEQ ID The primer sequence of Ampli1-RaB-R5 is shown in SEQ ID NO.12, the primer sequence of Ampli1-RaB-R5 is shown in SEQ ID NO.13, the primer sequence of Ampli1-RaB-R6 is shown in SEQ ID NO.14, the primer sequence of Ampli1-RaB-R7 is shown in SEQ ID NO.15, the primer sequence of Ampli1-RaB-R8 is shown in SEQ ID NO.16, the primer sequence of Ampli1-RaB-R9 is shown in SEQ ID NO.17, the primer sequence of Ampli1-RaB-R10 is shown in SEQ ID NO.18, the primer sequence of Ampli1-RaB-R11 is shown in SEQ ID NO.19, the primer sequence of Ampli1-RaB-R12 is shown in SEQ ID NO.20, the primer sequence of Ampli1-RaB-R13 is shown in SEQ ID NO.21, and the primer sequence of Ampli1-RaB-R14 is shown in SEQ ID NO. As shown in NO.22, after the reaction is carried out according to the PCR program, magnetic beads are used for sorting and purification to obtain the cDNA molecules of rabbit TCR and BCR after the first round of enrichment.

[0008] In some embodiments, the second round of enrichment includes: taking the product of the first round of enrichment as a template, combining the TCR and BCR primers enriched in the second round as a mixture, and preparing a PCR reaction solution for the second round of enrichment, wherein the Ampli-F primer sequence in the TCR primer mixture in S6 is shown in SEQ ID NO.4, the Ampli2-RaT-R1 primer sequence is shown in SEQ ID NO.23, the Ampli2-RaT-R2 primer sequence is shown in SEQ ID NO.24, the Ampli2-RaT-R3 primer sequence is shown in SEQ ID NO.25, and the Ampli2-RaT-R4 primer sequence is shown in SEQ ID NO.26, the Ampli2-RaB-R1 primer sequence in the BCR primer mixture in S6 is shown in SEQ ID NO.27, the Ampli2-RaB-R2 primer sequence is shown in SEQ ID NO.28, the Ampli2-RaB-R3 primer sequence is shown in SEQ ID NO.29, and the Ampli2-RaB-R4 primer sequence is shown in SEQ ID NO. The primer sequence of Ampli2-RaB-R5 is shown in SEQ ID NO.30, the primer sequence of Ampli2-RaB-R5 is shown in SEQ ID NO.31, the primer sequence of Ampli2-RaB-R6 is shown in SEQ ID NO.32, the primer sequence of Ampli2-RaB-R7 is shown in SEQ ID NO.33, the primer sequence of Ampli2-RaB-R8 is shown in SEQ ID NO.34, the primer sequence of Ampli2-RaB-R9 is shown in SEQ ID NO.35, the primer sequence of Ampli2-RaB-R10 is shown in SEQ ID NO.36, the primer sequence of Ampli2-RaB-R11 is shown in SEQ ID NO.37, the primer sequence of Ampli2-RaB-R12 is shown in SEQ ID NO.38, the primer sequence of Ampli2-RaB-R13 is shown in SEQ ID NO.39, and the primer sequence of Ampli2-RaB-R14 is shown in SEQ ID NO. As shown in NO.40, after the reaction is carried out according to the PCR program, magnetic beads are used for sorting and purification to obtain the cDNA molecules of rabbit TCR and BCR after the second round of enrichment.

[0009] In some embodiments, the construction of the single-cell immune repertoire library in S7 includes: using a library construction kit based on Tn5 transposase to simultaneously construct TCR and BCR libraries, Tn5 breaks TCR and BCR cDNA molecules and adds adapter sequences at both ends of them, and after transposition is completed, index PCR is performed, and the amplified library products are sorted and purified to obtain the single-cell TCR and BCR libraries.

[0010] The primer sequences described in the present invention are shown in the table below.

[0011]

[0012]

[0013]

[0014]

[0015] 1. Compared with the prior art, the present invention has the following beneficial effects: the present invention is the first to design a set of primers targeting the C region gene sequences of rabbit TCR and BCR / IG, and provides the first solution for the construction of rabbit single cell immune repertoire library; 2. The present invention is the first to use primers targeting TCR and BCR to simultaneously construct a mixed library of TCR and BCR, which significantly improves the efficiency of immune group library construction; 3. The present invention can simultaneously construct a single-cell immune repertoire library for 500-30,000 cells in a rabbit sample at single-cell resolution; 4. The present invention uses only a few primers targeting the C region to capture all TCR and BCR molecules in rabbit cells, avoiding a complex multiplex PCR system and a PCR system with a wide variety of primers; 5. The present invention can simultaneously obtain the α chain and β chain of the TCR of a single T cell in a rabbit sample, as well as the light chain and heavy chain of the BCR of a single B cell, thereby detecting the pairing information of two different chains in a single cell, which is difficult to achieve with traditional bulk methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] Figure 1 It is the technical route and principle diagram of the present invention.

[0017] Figure 2 This is the quality inspection picture of rabbit 5'cDNA.

[0018] Figure 3 This is the quality inspection picture of the rabbit TCR and BCR mixed library. DETAILED DESCRIPTION

[0019] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] The present invention is based on a droplet microfluidics system to generate oil-in-water droplets that only encapsulate a single cell. By enriching and amplifying the cDNA after reverse transcription of the droplet, the immune repertoire information of the rabbit TCR and BCR in the single cell can be obtained.

[0021] Figure 1 The technical route and principle diagram of the present invention are shown in Figure 1. Rabbit single cell suspension is matched with a customized microfluidic chip and oil-water system to produce a large number of oil-in-water reaction droplets encapsulating single rabbit cells and gel microspheres. After breaking the droplets and purifying the cDNA molecules, the special TCR and BCR primer combination of the present invention is used to enrich the rabbit immune repertoire TCR and BCR cDNA molecules from the whole genome cDNA after two rounds of PCR enrichment, and a second-generation sequencing library is constructed.

[0022] To achieve the above purpose: the present invention adopts the following technical solutions: When the quality of the single cell suspension is qualified, the microfluidics-based rabbit single cell immune repertoire construction method comprises the following steps: 1. Primer Design Based on the existing methodological gaps in the relevant field, the present invention designs a set of primer sequences targeting the C region genes in rabbit TCR (T cell receptor) and BCR / IG (B cell receptor / antibody), and the sequences are shown in SEQ ID NO.5-40.

[0023] 2. Primer Synthesis The primers needed are synthesized by a primer company, and the primers for rabbit TCR and BCR enrichment are mixed together when used. The mixed primers can be directly used during amplification.

[0024] 3. Preparation of Rabbit Single Cell Suspension The main sample types targeted by this technology are samples rich in immune cells, including rabbit PBMC and rabbit spleen. When rabbit PBMC (rabbit peripheral blood mononuclear cells) is used as a sample, the recovery process of rabbit PBMC is as follows: 1) Commercially available frozen PBMC samples were stored in liquid nitrogen; 2) Before use, thaw PBMC cells in a 37°C water bath; 3) Wash PBMC cells three times with warm PBS; 4) Finally, resuspend about 5 million PBMC cells in 3 mL PBS; 5) Take out an appropriate amount of cells, with a total of 500-30,000 cells, add BSA (SIGMA, Catalog No.: A1933-25G) with a final concentration of 0.04%, and add OptiPrep solution (SIAMA, D1556) with a final concentration of 9%; 6) Gently pipette the cell phase to mix until it is thoroughly mixed and a single-cell suspension is formed.

[0025] 4. Preparation of reverse transcription system Prepare the reverse transcription phase according to the following table:

[0026] 5. Preparation of Molecular Labeling Microspheres The microspheres are connected to oligonucleotide oligo sequences with different combination molecular tags, and the end of the sequence carries a modified sequence of rGrGrG for template conversion. The microspheres used in the present invention can be conventional in the art, such as polyacrylamide microspheres with specific oligonucleotide sequences (the preparation method refers to patent PCT / CN2021 / 129694), or double-layer microspheres with oligonucleotide sequences for single-cell sequencing (the preparation method refers to patent PCT / CN2021 / 12970).

[0027] 6. Preparation of Oil for Droplet Generation The generated oil (Bio-Rad, 1863005) can form oil-in-water droplets when combined with water. Theoretically, each droplet contains at most one microsphere and cell.

[0028] 7. Preparation of Single-cell Water-in-Oil Droplets and Reverse Transcription 1) Preparation of single-cell water-in-oil droplets: a. Add 50 μL MobiCube 5' transcriptome microspheres (Cat. No. S060500101 or S060500101) into well B of MobiCube Biochip B; b. Add 45 μL of rabbit single cell suspension to well C of chip B in the same way; c. Add 80 μL of reverse transcription phase to well D of chip B; d. Finally, add 150 μL of droplet generation oil into the sample well E of chip B; e. Be careful not to introduce bubbles during all sample addition operations. Use MobiNOva-100 library construction system of Mozobio to run relevant programs. It takes about 6 minutes to complete the generation of water-in-oil droplets. f. Carefully pipette approximately 140 μL of the droplet using a wide-mouth pipette tip and transfer to a low-binding 1.5 mL centrifuge tube.

[0029] 2) Droplet reverse transcription The droplets in the 1.5 mL centrifuge tube were pretreated with UV to fully release the oligo on the microspheres. The UV pretreatment process lasted about 5 minutes. After the treatment, 140 μL of the droplets were carefully divided into two 200 μL PCR tubes and reverse transcription reactions were performed on a PCR instrument. The procedure was as follows:

[0030] 8. Demulsification and purification of droplets 1) After reverse transcription is completed, add 100 μL of recovery agent (1% PFO, SIGMA, 370533) to the droplets in each 200 μL PCR tube, and mix thoroughly by inversion to break the emulsion droplets and release the contents in the droplets. The oil and water phases will separate, and the water phase containing the reverse transcribed cDNA will be located on the upper layer of the oil phase; 2) Transfer all the upper aqueous phases in the two PCR tubes to the same centrifugal filter tube (SIGMA, CLS8162). The interface between the aqueous phase and the oil phase contains a large number of gel microspheres, which need to be removed with the filter tube; 3) Centrifugation, 10,000 g, 5 min; 4) Repeat step 3) until all the liquid on the filter membrane is filtered to the bottom of the tube; 5) Use SPRIselect magnetic beads to purify the aqueous phase collected in the centrifuge tube 1.2x, elute the cDNA with 54 μL ultrapure water to obtain the purified cDNA product.

[0031] 9. cDNA pre-amplification and purification 1) cDNA pre-amplification a. Prepare the PCR reaction system according to the following table:

[0032] b. The PCR procedure is as follows:

[0033] Steps 2-4 are cycle steps. The specific number of cycles depends on the tissue type. Please refer to the following table:

[0034] 2) Purification of cDNA pre-amplification products The pre-amplified cDNA product was recovered at 0.6x using SPRIselect magnetic beads, and finally the cDNA molecules were eluted with 20 μL of ultrapure water to obtain 20 μL of purified cDNA product, which can be used as a template for the subsequent enrichment of rabbit TCR and BCR related cDNA molecules and the construction of immune repertoire library.

[0035] 10. Enrichment of Rabbit TCR and BCR Molecules 1) Using the cDNA product recovered in step 9.2) as a template, perform the first round of enrichment of rabbit TCR and BCR molecules. The enrichment process and procedures are as follows: a. Take 1 μL cDNA as template and prepare the first round of rabbit TCR and BCR enrichment system according to the following table:

[0036]

[0037] The PCR program is as follows:

[0038] Steps 2-4 are cycling steps, and the number of cycles for the first round of enrichment of rabbit TCR and BCR is 12.

[0039] b. The rabbit TCR and BCR products after the first round of enrichment were sorted by magnetic beads, and SPRIselect magnetic beads were used for 0.5x and 0.8x sorting. Finally, 30 μL ultrapure water was used to elute the rabbit TCR and BCR enriched and purified products.

[0040] 2) Using the cDNA product recovered in step 10.2) b as a template, a second round of enrichment of rabbit TCR and BCR molecules was performed. The enrichment process and procedures are as follows: a. Take 30 μL of cDNA recovery product as a template and prepare the second round of rabbit TCR and BCR enrichment system according to the following table:

[0041]

[0042] The PCR program was the same as that of the first round of enrichment of rabbit TCR and BCR, and the number of cycles for the second round of enrichment of rabbit TCR and BCR was 10.

[0043] b. The rabbit TCR and BCR products after the second round of enrichment were sorted by magnetic beads, and SPRIselect magnetic beads were used for 0.5x and 0.8x sorting. Finally, 30 μL ultrapure water was used to elute the rabbit TCR and BCR enriched and purified products.

[0044] 11. Construction of Rabbit Single Cell Immune Repertoire Library The rabbit TCR and BCR cDNA products after the second round of enrichment and purification in step 10 (2) b were used to construct a library. The library was constructed using Hieff NGS® Fast Tagment DNA Library Prep Kit for Illumina® (for 50ng) (Yisheng, 12207ES24). Tn5 transposase was mainly used to shear the enriched cDNA, and sequencing adapters were added at the same time. Finally, PCR enrichment was performed to quickly obtain a library compatible with the Illumina sequencing platform.

[0045] Embodiment 1 Using rabbit PBMC samples as starting materials, the construction of a rabbit single cell immune repertoire library based on microfluidics was carried out, including the following steps: 1. Design rabbit TCR and BCR enrichment related primers, the sequences are shown in SEQ ID NO.5-40.

[0046] The present invention compares the existing optimal solution with the suboptimal solution, which shows that the present invention is more efficient, has better specificity and higher sensitivity.

[0047] The suboptimal solution is a solution designed and tested by the inventors in the early development of the rabbit single-cell immune repertoire. The performance indicators of the TCR matching rate and BCR (IG) matching rate of the suboptimal solution are not high enough, and there are no relevant patents for the rabbit single-cell immune repertoire on the market. Therefore, the inventors invented the preferred solution described in this application through updating and iteration, and the primer sequences of the suboptimal solution are shown in SEQ ID NO.41-68.

[0048] 2. Select the required primers and have them synthesized by a primer company; 3. Preparation of Rabbit Single Cell Suspension 1) Commercially available frozen PBMC samples were stored in liquid nitrogen; 2) Before use, thaw PBMC cells in a 37°C water bath; 3) Wash PBMC cells three times with warm PBS; 4) Finally, resuspend about 5 million PBMC cells in 3 mL PBS; 5) Take out an appropriate amount of cells, with a total of 10,000 cells, add BSA (SIGMA, Catalog No.: A1933-25G) with a final concentration of 0.04%, and add OptiPrep solution (SIAMA, D1556) with a final concentration of 9%; 6) Gently pipette the cell phase to mix until it is thoroughly mixed and a single-cell suspension is formed.

[0049] 4. Preparation of reverse transcription system Prepare the reverse transcription phase according to the following table:

[0050] 5. Preparation of molecular labeling microspheres The microspheres are connected to oligonucleotide oligo sequences with different combination molecular tags, and the end of the sequence carries a modified sequence of rGrGrG for template conversion. The microspheres used in the present invention can be conventional in the art, such as polyacrylamide microspheres with specific oligonucleotide sequences (reference patent PCT / CN2021 / 129694), or double-layer microspheres with oligonucleotide sequences for single-cell sequencing (reference patent PCT / CN2021 / 12970).

[0051] 6. Prepare the droplet generation oil The oil (Bio-Rad, 1863005) can form water-in-oil droplets when combined with water, and each droplet theoretically contains at most one microsphere and cell.

[0052] 7. Preparation of single-cell water-in-oil droplets and reverse transcription 1) Preparation of single-cell water-in-oil droplets: a. Add 50 μL of MobiCube 5' transcriptome microspheres into well B of MobiCube Biochip B; b. Add 45 μL of rabbit single cell suspension to well C of chip B in the same way; c. Add 80 μL of reverse transcription phase to well D of chip B; d. Finally, add 150 μL of droplet generation oil into the sample well E of chip B; e. Be careful not to introduce bubbles during all sample addition operations. Use MobiNOva-100 library construction system of Mozobio to run relevant programs. It takes about 6 minutes to complete the generation of water-in-oil droplets. f. Carefully pipette approximately 140 μL of the droplet using a wide-mouth pipette tip and transfer to a low-binding 1.5 mL centrifuge tube.

[0053] 2) Droplet reverse transcription: Place the droplets in the 1.5 mL centrifuge tube into the UV pretreatment device of MoZhuo Biotechnology for UV treatment to fully release the oligo on the microspheres. The UV pretreatment process takes about 5 minutes. After treatment, carefully divide the 140 μL droplets into two 200 μL PCR tubes and perform reverse transcription reaction on the PCR instrument. The procedure is as follows:

[0054] 8. Demulsification and purification of droplets 1) After reverse transcription is completed, add 100 μL of recovery agent (1% PFO, SIGMA, 370533) to the droplets in each 200 μL PCR tube, and mix thoroughly by inversion to break the emulsion droplets and release the contents in the droplets. The oil and water phases will separate, and the water phase containing the reverse transcribed cDNA will be located on the upper layer of the oil phase; 2) Transfer all the upper aqueous phases in the two PCR tubes to the same centrifugal filter tube (SIGMA, CLS8162). The interface between the aqueous phase and the oil phase contains a large number of gel microspheres, which need to be removed with the filter tube; 3) Centrifugation, 10,000 g, 5 min; 4) Repeat step 3) until all the liquid on the filter membrane is filtered to the bottom of the tube; 5) Use SPRIselect magnetic beads to purify the aqueous phase collected in the centrifuge tube 1.2x, elute the cDNA with 54 μL ultrapure water to obtain the purified cDNA product.

[0055] 9. cDNA Pre-amplification and Purification 1) cDNA pre-amplification a. Prepare the PCR reaction system according to the following table:

[0056] b. The PCR procedure is as follows:

[0057] Steps 2-4 are cycle steps. The specific number of cycles depends on the tissue type. Please refer to the following table:

[0058] This time it was a PBMC sample, and the number of amplification cycles was 14.

[0059] 2) Purification of cDNA pre-amplification products The pre-amplified cDNA product was recovered at 0.6x using SPRIselect magnetic beads, and the cDNA molecules were eluted with 20 μL of ultrapure water to obtain 20 μL of purified cDNA product with a concentration of 10.3 ng / μL. Figure 2 This is the Q-sep 100 quality inspection peak diagram of the corresponding pre-amplified cDNA. The quality inspection results show that the cDNA fragment size is normal.

[0060] 10. Enrichment of Rabbit TCR and BCR Molecules 1) Using the cDNA product obtained in step 2) of step 9 as a template, perform the first round of enrichment of rabbit TCR and BCR molecules. The enrichment process and procedures are as follows: a. Take 1 μL cDNA as template and prepare the first round of TCR and BCR enrichment system according to the following table:

[0061]

[0062] Among them, the system of the first round of TCR and BCR enrichment of the suboptimal solution compared with the present invention is as follows:

[0063] The PCR program is as follows:

[0064] Steps 2-4 are cycling steps, and the number of cycles for the first round of enrichment of rabbit TCR and BCR is 12.

[0065] b. The rabbit TCR and BCR products after the first round of enrichment were sorted by magnetic beads, and SPRIselect magnetic beads were used for 0.5x and 0.8x sorting. Finally, 30 μL ultrapure water was used to elute the rabbit TCR and BCR enriched and purified products. The concentration of the first round of enrichment products is as follows:

[0066] 2) Using the enriched cDNA product recovered in step 1) b in step 10 as a template, a second round of enrichment of rabbit TCR and BCR molecules was performed. The enrichment process and procedures are as follows: a. Take 30 μL of cDNA recovery product as a template and prepare the second round of rabbit TCR and BCR enrichment system according to the following table:

[0067]

[0068] Among them, the system of the second round of TCR and BCR enrichment of the suboptimal solution compared with the present invention is as follows:

[0069] The PCR program was the same as that of the first round of rabbit TCR and BCR enrichment, and the number of cycles for the second round of rabbit TCR and BCR enrichment was 10.

[0070] b. The rabbit TCR and BCR products after the second round of enrichment were sorted by magnetic beads, and SPRIselect magnetic beads were used for 0.5x and 0.8x sorting. Finally, 30 μL ultrapure water was used to elute the rabbit TCR and BCR enriched and purified products. The concentration of the second round of enrichment products is as follows:

[0071] 11. Construction of Rabbit Single Cell Immune Repertoire Library The rabbit TCR and BCR cDNA products after the second round of enrichment and purification in step 2) of step 10 were constructed using Hieff NGS® Fast Tagment DNA Library Prep Kit for Illumina® (for 50 ng) (Yisheng, 12207ES24). Tn5 transposase was mainly used to shear the enriched cDNA, and sequencing adapters were added at the same time. Finally, PCR enrichment was performed to quickly obtain a library compatible with the Illumina sequencing platform. The index PCR product was sorted with SPRIselect magnetic beads at 0.5x and 0.65x fragments, and the DNA was eluted with 20 μL ultrapure water to obtain the rabbit single cell immune group library. The quality inspection peak diagram of the rabbit TCR and BCR mixed library is attached. Figure 3 The peak shape of the mixed TCR and BCR library was normal, indicating a good enrichment effect. The library was sequenced on the Illumina NOvaSEQ 6000 platform.

[0072]

[0073] 12. Analysis of Rabbit Immune Repertoire Sequencing Results 1) The sequencing results of the TCR and BCR libraries of the rabbit PBMC were analyzed, and the primer combination results of the present invention were compared with the primer combination results of the suboptimal solution. The main comparison results are shown in the following table:

[0074] The results show that the present invention provides a faster and more efficient strategy for the simultaneous construction of rabbit single cell immune repertoire TCR and BCR libraries. Compared with the traditional method of separately constructing TCR library and BCR library, the present invention realizes the simultaneous construction of TCR library and BCR library in the same reaction, thereby improving the efficiency of library construction. Compared with the traditional library construction strategy based on DNA fragmentation, end repair, linker connection, and PCR enrichment, the present invention adopts a library construction strategy based on Tn5, which only requires transposition and PCR enrichment to efficiently complete library construction, greatly improving the efficiency of library construction.

[0075] Using the optimal rabbit immune repertoire enrichment primer combination designed by the present invention, and by matching MobiNOva-100 library construction system and MobiCube high-throughput single-cell 5' transcriptome reagent set of Mozhuo Bio, the present invention obtains the rabbit single-cell immune repertoire library and the corresponding effective sequencing results for the first time on the basis of the existing methods. The present invention realizes the simultaneous construction of TCR and BCR libraries, and can split the TCR and BCR data in the TCR and BCR mixed library respectively. This is also the first time in the existing methods in the relevant field that the primer combination and corresponding solution can be used to obtain rabbit single-cell immune repertoire information.

[0076] At the same time, the present invention was compared with the suboptimal primer combination scheme. Approximately 5,000 cells were captured in this experiment. 1,104 rabbit T cells were captured in the TCR library of the present invention, and 2,602 rabbit B cells were captured in the BCR library. The 651 TCR cells and 1,872 BCR cells captured in the TCR and BCR libraries in the suboptimal scheme were significantly lower than those of the present invention.

[0077] The enrichment of the rabbit TCR and BCR libraries was good, and >70% of the reads in the TCR and BCR libraries could be mapped to any V(D)J gene, while only 40-47% of the reads in the suboptimal solution could be mapped to any V(D)J gene. This shows that the specificity of the C region primers targeting rabbit TCR and BCR in the present invention is significantly higher than that of the suboptimal solution primers, and can more specifically enrich cDNA molecules related to rabbit TCR and BCR.

[0078] In addition, the present invention is able to capture the pairable double-stranded information across VJ segments in 65% of all captured T cells, while the double-stranded pairing rate across VJ segments in T cells of the suboptimal solution is 43%.

[0079] Similarly, the present invention can capture the pairable double-chain information of the VJ fragments in 82% of all captured B cells, while the double-chain pairing rate across VJ fragments in B cells of the suboptimal solution is 51%. This result shows that the present invention is a better or even the best solution known so far, achieving single-cell resolution, and can explore the pairing information of the α chain and β chain (or heavy and light chain) in most captured single T cells or B cells, and the effect is better than other solutions. Preferably, through MobiNova library construction system of Mozo, the present invention can obtain up to tens of thousands of rabbit-derived T cells or B cells with double-chain pairing information in a very short time.

[0080] It should be noted that our results show that the percentage of cells containing pairable double-stranded proteins across VJ segments in our TCR or BCR library is still not close to 100%, which means that the double-stranded pairing information of a small number of captured cells cannot be detected in single cells. In the future, it may be necessary to adjust the primer combination, primer concentration and PCR conditions used in the present invention to improve the pairing rate.

[0081] The above is a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for simultaneously constructing a rabbit single cell immune repertoire TCR and BCR library based on droplet microfluidics, characterized in that: The construction method comprises the following steps: S1. Synthesize primer sequences targeting rabbit TCR and BCR / IG, the primer sequences are shown in SEQ ID NO.5-40; S2. Preparation of single cell suspension; S3. Preparation and reverse transcription of single-cell oil-in-water droplets; S4. Demulsification and cDNA pre-amplification; S5. Rabbit TCR and BCR / IG related cDNA molecules were simultaneously enriched in the first round; S6. Rabbit TCR and BCR / IG related cDNA molecules were simultaneously enriched in the second round; S7. Construction of a more rapid rabbit single cell immune repertoire TCR and BCR mixed library based on Tn5 transposase.

2. The construction method according to claim 1, characterized in that: The primer sequences for the first round of enrichment are shown in SEQ ID NO.4 and SEQ ID NO.5-22, and the primer sequences for the second round of enrichment are shown in SEQ ID NO.4 and SEQ ID NO.23-40.

3. The construction method according to claim 2, characterized in that: The oligo-dT primer sequence in the reverse transcription of S3 is shown in SEQ ID NO.

1.

4. The construction method according to claim 3, characterized in that: The demulsification and cDNA pre-amplification in S4 include: using a demulsifier to demulsify the droplets after reverse transcription in S3 to obtain a recovered product, and performing PCR pre-amplification on the recovered product using a cDNA pre-amplification primer sequence, wherein the cDNA-Amp-F primer sequence in the cDNA pre-amplification is shown in SEQ ID NO.2, and the cDNA-Amp-R primer sequence is shown in SEQ ID NO.

3.

5. The construction method according to claim 4, characterized in that: The first round of enrichment includes: taking the cDNA in S4 as a template, combining the TCR and BCR primers enriched in the first round as a mixture, and preparing a first round of enrichment PCR reaction solution, the TCR primers in S5 include the Ampli-F primer sequence as shown in SEQ ID NO.4, the Ampli1-RaT-R1 primer sequence as shown in SEQ ID NO.5, the Ampli1-RaT-R2 primer sequence as shown in SEQ ID NO.6, the Ampli1-RaT-R3 primer sequence as shown in SEQ ID NO.7, and the Ampli1-RaT-R4 primer sequence as shown in SEQ ID NO.8, the BCR primers in S5 include the Ampli1-RaB-R1 primer sequence as shown in SEQ ID NO.9, the Ampli1-RaB-R2 primer sequence as shown in SEQ ID NO.10, the Ampli1-RaB-R3 primer sequence as shown in SEQ ID NO.11, and the Ampli1-RaB-R4 primer sequence as shown in SEQ ID NO. The primer sequence of Ampli1-RaB-R5 is shown in SEQ ID NO.12, the primer sequence of Ampli1-RaB-R5 is shown in SEQ ID NO.13, the primer sequence of Ampli1-RaB-R6 is shown in SEQ ID NO.14, the primer sequence of Ampli1-RaB-R7 is shown in SEQ ID NO.15, the primer sequence of Ampli1-RaB-R8 is shown in SEQ ID NO.16, the primer sequence of Ampli1-RaB-R9 is shown in SEQ ID NO.17, the primer sequence of Ampli1-RaB-R10 is shown in SEQ ID NO.18, the primer sequence of Ampli1-RaB-R11 is shown in SEQ ID NO.19, the primer sequence of Ampli1-RaB-R12 is shown in SEQ ID NO.20, the primer sequence of Ampli1-RaB-R13 is shown in SEQ ID NO.21, and the primer sequence of Ampli1-RaB-R14 is shown in SEQ ID NO. As shown in NO.22, after the reaction is carried out according to the PCR program, magnetic beads are used for sorting and purification to obtain the cDNA molecules of rabbit TCR and BCR after the first round of enrichment.

6. The construction method according to claim 5, characterized in that: The second round of enrichment includes: taking the product of the first round of enrichment as a template, combining it with the TCR and BCR primers enriched in the second round as a mixture, and preparing a PCR reaction solution for the second round of enrichment, wherein the TCR primers in S6 include the Ampli-F primer sequence as shown in SEQ ID NO.4, the Ampli2-RaT-R1 primer sequence as shown in SEQ ID NO.23, the Ampli2-RaT-R2 primer sequence as shown in SEQ ID NO.24, the Ampli2-RaT-R3 primer sequence as shown in SEQ ID NO.25, and the Ampli2-RaT-R4 primer sequence as shown in SEQ ID NO.26, and the BCR primers in S6 include the Ampli2-RaB-R1 primer sequence as shown in SEQ ID NO.27, the Ampli2-RaB-R2 primer sequence as shown in SEQ ID NO.28, the Ampli2-RaB-R3 primer sequence as shown in SEQ ID NO.29, and the Ampli2-RaB-R4 primer sequence as shown in SEQ ID NO. The primer sequence of Ampli2-RaB-R5 is shown in SEQ ID NO.30, the primer sequence of Ampli2-RaB-R5 is shown in SEQ ID NO.31, the primer sequence of Ampli2-RaB-R6 is shown in SEQ ID NO.32, the primer sequence of Ampli2-RaB-R7 is shown in SEQ ID NO.33, the primer sequence of Ampli2-RaB-R8 is shown in SEQ ID NO.34, the primer sequence of Ampli2-RaB-R9 is shown in SEQ ID NO.35, the primer sequence of Ampli2-RaB-R10 is shown in SEQ ID NO.36, the primer sequence of Ampli2-RaB-R11 is shown in SEQ ID NO.37, the primer sequence of Ampli2-RaB-R12 is shown in SEQ ID NO.38, the primer sequence of Ampli2-RaB-R13 is shown in SEQ ID NO.39, and the primer sequence of Ampli2-RaB-R14 is shown in SEQ ID NO. As shown in NO.40, after the reaction is carried out according to the PCR program, magnetic beads are used for sorting and purification to obtain the cDNA molecules of rabbit TCR and BCR after the second round of enrichment.

7. The construction method according to claim 6, characterized in that: The S3 was carried out using the MobiNova-100 library construction system of MobiBio and the MobiCube high-throughput single-cell 5' transcriptome reagent kit v2.0 kit.

8. The construction method according to claim 7, characterized in that: The rabbit single cell immune repertoire TCR and BCR mixed library in the S7 is constructed using a transposase-based library construction kit. Compared with the traditional library construction method based on breaking enzymes and linker ligation, the combined library of TCR and BCR can be obtained more quickly and efficiently.

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