A method for constructing a spatial transcriptome sequencing library based on evenly distributed combinatorially encoded microspheres

Abstract

The present invention discloses a method for constructing a spatial transcriptome sequencing library based on equally divided combination coding microspheres, comprising the following steps: design and preparation of DNA-encoded microspheres based on the concept of equally divided combination; design of transcriptome sequencing library construction (SCDB-seq) based on SCDB microspheres; design and implementation of spatial transcriptome sequencing based on SCDB-seq. This method can perform selective, highly multiplexed medium-throughput (50 to 1000 samples) spatial transcriptome sequencing library construction on microregions on fresh frozen sample sections and formalin-fixed paraffin-embedded sample sections at single-cell resolution (~10μm), and can selectively obtain gene expression profiles of multiple specific microregion samples in tissue sections; the experimental operation amount and reagent cost of this method are only ~1% and ~13% of existing similar methods, showing good control of manpower and reagent costs.

Description

Technical Field

[0001] The present invention relates to a method for constructing an NGS sequencing cDNA library for single-cell transcriptome sequencing and spatial transcriptome sequencing, and in particular to a method for constructing a transcriptome sequencing library with medium detection throughput (50 to 1000 cells) and high multiplexing capability that can be adapted to tissue section micro-area sample collection methods such as laser capture microdissection (LCM), laser-induced forward transfer (LIFT), and micro-area sampling. Background Art

[0002] NGS-based spatially resolved transcriptome technology uses primers with oligo-dT to capture mRNA in the target microregion, and then applies a certain RNA-seq protocol for reverse transcription and cDNA amplification to obtain a cDNA library that meets sequencing requirements. Existing NGS-based spatially resolved transcriptomics technologies often directly apply cDNA library construction methods or improved versions of scRNA-seq protocols with comparable detection throughput. For example, in high-throughput methods based on in situ capture, ST and HDST use the library construction and sequencing protocol in InDrop. This involves linear amplification of cDNA through IVT after synthesis, followed by secondary reverse transcription and PCR amplification to generate sequencing libraries, enabling highly parallel and highly sensitive detection of low-abundance transcripts. Slide-seq and DBiT-seq use the protocol in Drop-Seq, a version based on the Smart-seq2 protocol improved for high-throughput detection. Template switching and two rounds of PCR amplification are still performed during reverse transcription, but after Tn5 transposase disruption, only the 3'-end DNA fragments are amplified to retain the DNA barcode and UMI sequences. In spatially resolved transcriptomics methods based on microdissection, Geo-seq and LCM-seq directly use the reverse transcription and cDNA library construction protocols of Smart-seq2 to sequence the full-length mRNA molecules within the target microregion.

[0003] With the development of various microdissection and microarea sampling technologies, the time required to sample a single target region has been continuously shortened, and the degree of automation continues to increase. This has the potential to enable serial sampling of hundreds or even thousands of target microareas within a set of tissue sections. However, currently, such technologies rely solely on Smart-seq2 for sequencing library construction. Smart-seq2 cannot use capture primers with DNA barcodes and can only perform specific labeling using different index sequences during PCR amplification and library construction. Therefore, Smart-seq2 is a low-throughput, low-parallelism protocol for full-length transcriptome sequencing (mostly targeting a few or dozens of microarea samples). During library construction, individual microarea samples cannot be mixed for parallel library construction and sequencing after reverse transcription. This results in experimental workload and reagent consumption proportional to the number of microarea samples collected. This limits the ability of microdissection-based spatially resolved transcriptomics methods to perform medium-throughput (50–1000 samples) and high-parallelism gene expression profiling of target regions within tissue sections, hindering the application of such technologies to elucidate region-specific gene expression profiles and collective cellular behavior within biological tissues. Summary of the Invention

[0004] Purpose of the invention: The purpose of the present invention is to provide a method for constructing a spatial transcriptome sequencing library based on equally divided combination coding microspheres with good detection effect, low cost, selectivity and high multiplexing.

[0005] Technical solution: The method for constructing a spatial transcriptome sequencing library based on equally divided combination coding microspheres comprises the following steps:

[0006] (1) Design and preparation of SCDB-based microspheres;

[0007] (2) Design and use of SCDB-seq;

[0008] (3) Design and implementation of spatial transcriptome sequencing based on SCDB-seq.

[0009] Furthermore, the design method of step (1) is as follows: two primers with specific sequence designs: primer 1-N (1≤N≤8) and primer 2-M (1≤M≤12) are used to prepare DNA barcode primers on the surface of the microspheres using an equal distribution combination preparation method. A certain amount of coded microspheres are divided into 8 parts and placed in centrifuge tubes and named "1-N" (1≤N≤8), and the corresponding primer 1-N is added to the centrifuge tube for the first round of encoding. Subsequently, the microspheres in the 8 centrifuge tubes are divided into a 96-well PCR plate in a specific order (centrifuge tubes 1-1 to 1-8 correspond to rows A to H on the 96-well PCR plate) and the corresponding primer 2-M is added for the second round of encoding, and finally 96 types of coded microspheres with unique DNA barcode combinations are generated.

[0010] Furthermore, the design method of step (1) is as follows: 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) is used to convert the carboxyl groups on the surface of the microsphere into O-acylisourea intermediates that can react with amino groups. This intermediate can quickly form an amide bond with primer 1-N modified with an amino group at the 5' end, thereby covalently fixing primer 1-N to the surface of the microsphere. With the special primer design, the 3' end of primer 2-M can produce reverse complementarity with the 3' end of primer 1-N. Subsequently, under the action of isothermal amplification DNA polymerase, the reverse complement of the sequence in primer 2-M is extended to the 3' end of primer 1-N, thereby completing the preparation of the microsphere surface encoding primer.

[0011] Furthermore, the sequence design (5'→3') of the primer 1-N is: 1) PCR primer (PCR Primer), used to amplify the cDNA library by PCR; 2) DNA bar 1-N (Barcode1-N), a specific barcode sequence that plays a DNA coding role, a total of 8 types; 3) a linker sequence (Adapter), used to reverse complement with the 3' end of primer 2-M; the sequence design (5'→3') of the primer 2-M is: 1) poly-dA, used to generate poly-dT with mRNA capture function; 2) UMI*, a sequence that plays a specific molecular recognition role; 3) DNA bar 2-M* (Barcode2-M*), a specific coding sequence that plays a DNA coding role, a total of 12 types; 4) a reverse complementary linker sequence (Adapter*), used to reverse complement with the 3' end of primer 1-N.

[0012] Furthermore, the microspheres are monodisperse magnetic microspheres, the inner core of the microspheres is SiO2 or polystyrene, the outer core is covered with a layer of Fe3O4, and the surface is coated with a chemical modification layer modified to carboxyl groups.

[0013] Furthermore, the particle size of the microspheres is 0.1 μm to 100 μm, and microspheres with particle sizes of 200 μm, 1 μm, and 20 μm are used in the specific operation.

[0014] Furthermore, the design method of SCDB-seq in step (2) comprises the following steps:

[0015] 1) Use SCDB microspheres to capture mRNA in samples;

[0016] 2) reverse transcription of the captured mRNA molecules;

[0017] 3) Construction of a cDNA library suitable for next generation sequencing (NGS) by PCR amplification;

[0018] 4) Use purification magnetic beads to enrich the target cDNA fragment.

[0019] Furthermore, the spatial transcriptome sequencing based on SCDB-seq in step (3) is characterized by:

[0020] 1) The protocol used for fresh-frozen tissue sections added 0.5% Triton X-100 to permeabilize the cell membrane during mRNA capture, allowing mRNA to be fully released from the cells and captured by SCDB microspheres;

[0021] 2) In addition to adding 0.5% Triton X-100 to the experimental protocol applied to FFPE tissue sections, the Mg2+ heat treatment step, which is used to fragment mRNA, was omitted because the mRNA in FFPE tissue sections has already been degraded to a certain extent. In addition, 0.125 μg / μL proteinase K was added to the mRNA release and capture steps, and the incubation was performed at 60°C for 1 hour to fully remove the intermolecular cross-links in the FFPE tissue and release the mRNA molecules as much as possible.

[0022] The preferred solution is as follows:

[0023] The two primers used for synthesizing SCDB microspheres: Primer 1-N (1≤N≤8) and Primer 2-M (1≤M≤12) have special primer designs: (1) Primer 1-N sequence design (5'→3') is: 1) PCR primer (PCR Primer), used to amplify cDNA library by PCR; 2) DNA bar 1-N (Barcode1-N), a specific barcode sequence that plays a DNA coding role, a total of 8 types; 3) Linker sequence (Adapter), used to reverse complement with the 3' end of Primer 2-M; (2) Primer 2-M sequence design (5'→3') is: 1) poly-dA, used to generate poly-dT with mRNA capture function; 2) UMI*, a sequence that plays a specific molecular recognition role; 3) DNA bar 2-M* (Barcode2-M*), a specific coding sequence that plays a DNA coding role, a total of 12 types; 4) Reverse complementary linker sequence (Adapter*), used to reverse complement with Primer 1-N The 3' end is reverse complementary.

[0024] DNA encoding primers were prepared on the microsphere surface using primers 1-N and 2-M using an evenly divided combination preparation method. Specifically, a certain amount of encoded microspheres were divided equally into eight 1.5ml centrifuge tubes and placed in each tube, each designated "1-N" (1≤N≤8). The corresponding primers 1-N were added to each tube for the first round of encoding. Subsequently, the microspheres from the eight tubes were evenly divided into a 96-well PCR plate in a specific order (centrifuge tubes 1-1 to 1-8 correspond to rows A to H on a 96-well PCR plate) and the corresponding primers 2-M were added for the second round of encoding. Ultimately, 96 encoded microspheres with unique DNA encoding combinations were generated. The specific synthesis scheme is that the carboxyl groups on the surface of the microspheres are converted into O-acylisourea intermediates that can react with amino groups under the action of EDC. This intermediate can quickly form an amide bond with primer 1-N modified with an amino group at the 5' end, thereby covalently fixing primer 1-N on the surface of the microspheres. Subsequently, with the help of special primer design, the 3' end of primer 2-M can produce reverse complementarity with the 3' end of primer 1-N. Subsequently, under the action of isothermal amplification DNA polymerase, the reverse complementarity of the sequence in primer 2-M is extended to the 3' end of primer 1-N, thereby completing the preparation of the microsphere surface encoding primer.

[0025] The SCDB microsphere primers used to synthesize DNA encoding primers are designed as follows: 1) PCR primers (PCR Primers), used to amplify the cDNA library by PCR; 2) DNA barcodes 1-N (Barcode 1-N), eight specific barcode sequences that act as DNA encoding; 3) adapter sequences (Adapters), used to reverse complement the 3' end of primer 2-M; 4) UMIs, sequences that act as specific molecular recognition; 5) DNA barcodes 2-M (Barcode 2-M), twelve specific barcode sequences that act as DNA encoding; and 6) poly-dT, used to capture mRNA.

[0026] SCDB-seq is suitable for tissue section micro-area sample collection methods such as LCM, LIFT and micro-area sampling. The FF tissue sections and FFPE tissue sections to be analyzed are stained with hematoxylin-eosin (H&E), so that the cell nuclei in the tissue sections appear blue-purple and the cytoplasm is red. Based on the results of H&E staining, LIFT microdissection is performed on the target area on the tissue section, and the diameter of a single cutting point is about 20μm. The experimental protocol applied to fresh frozen tissue sections adds a 0.5% concentration of Triton X-100 to the mRNA capture link to permeabilize the cell membrane, so that the mRNA can be fully released from the cells and captured by SCDB microspheres. In addition to adding a 0.5% concentration of Triton X-100 to the experimental protocol applied to FFPE tissue sections, the Mg used to fragment the mRNA is eliminated because the mRNA in the FFPE tissue sections already has a certain degree of fragmentation. 2+ During the heat treatment step, proteinase K at a concentration of 0.125 μg / μL was added during the mRNA release and capture step and incubated at 60°C for 1 hour to fully remove the intermolecular cross-links in the FFPE tissue and release the mRNA molecules as much as possible.

[0027] The volumes of various solutions were calculated based on the number of pre-collected samples, n. On an electronic thermostated sample station precooled to 0°C, n + n / 5 portions of mRNA fragmentation mix (Fragmentation Mix) and template switch-reverse transcription mix (TS-RT Mix) were prepared. Fragmentation mix and a 10 mg / mL SCDB microsphere suspension were added to PCR tubes or 96-well plates, respectively, and mixed by vortexing. Microregions of target tissue were collected into the cap of the PCR tube using laser-induced forward transfer (LIFT) or microarea sampling. The cap was then tightly closed and the PCR tube inverted to allow the solution to immerse the target tissue, permeabilizing and releasing RNA molecules. The PCR tube or 96-well plate was then inverted in a thermostated metal bath and lysed according to the protocol. The tube was then briefly centrifuged to concentrate the solution at the bottom of the tube. Complete lysis of the target sample was observed microscopically. If the target sample has been completely lysed, remove the PCR tube or 96-well plate and add LCM TS-RT Mix to each well. Then, mix by shaking. Place the PCR tube or 96-well plate in a constant temperature metal bath and complete reverse transcription according to the procedure.

[0028] The PCR tube or 96-well plate is then shaken, and the microsphere suspension is pipetted into a PCR tube. After magnetic adsorption, a portion of the supernatant is removed. A pre-amplification PCR mix is ​​prepared, and the PCR tube or 96-well plate is then placed in a PCR thermal cycler for pre-amplification. The cDNA library is then purified and concentrated using DNA purification magnetic beads.

[0029] Prepare the PCR amplification library mix and add it to the PCR tubes mentioned above. Place the tubes in a PCR thermal cycler and proceed with PCR amplification to construct the sequencing library. Purify and concentrate the cDNA library using DNA purification magnetic beads. Finally, perform a Qubit assay on the cDNA library and perform Aligent electrophoresis. If confirmed, send the library for testing.

[0030] The present invention is directed to the problem that the DNA barcode sequence of the DNA encoding (Split-Pool DNA barcode, SPDB) microspheres prepared by the mixing-equal division method is in an unknown state and needs to be decoded in some way for spatial resolution transcriptome sequencing. The present invention proposes a SCDB microsphere preparation scheme, by continuously dividing the microspheres and connecting different and known DNA barcodes to obtain a variety of encoding microspheres. Unlike SPDB microspheres, the DNA barcode sequence of the encoding microspheres prepared by this method is known. When used, the correspondence between the encoding microspheres used and the sample numbers is recorded to correspond the mRNA library obtained by sequencing to its spatial position information, thereby completing spatial resolution transcriptome sequencing. In order to ensure that RNA-seq is highly sensitive and accurate for mRNA molecules in microtissue samples or RNA degradation samples, such as FFPE tissues, the present invention is optimized on the basis of the Smart-3SEQ scheme to adapt it to SCDB microspheres, and is referred to as SCDB-seq. Comparing SCDB-seq with Smart-seq2 proved that this scheme can effectively detect the transcriptome within micro-area samples and has multiplexing capabilities for detecting multiple samples. It eliminates the need for tedious operations during the experiment and significantly reduces reagent costs.

[0031] Beneficial effects: Compared with the prior art, the present invention has the following advantages:

[0032] 1. This method improves upon the widely used split-pool strategy for preparing DNA-encoded microspheres, proposing a split-combinatorial approach. Based on this concept, 96 types of SCDB microspheres were designed and implemented. Unlike DNA-encoded microspheres prepared using the split-pool strategy, SCDB microspheres are loaded into a 96-well PCR plate. The DNA encoding sequence of each SCDB microsphere in each well is known and identical, corresponding to the encoding sequence of the microwell.

[0033] 2. This method fully considers the economic efficiency of experimental materials and reagents when designing the experimental plan, in order to obtain a transcriptome sequencing library construction method with excellent detection performance and low experimental cost. During the preparation of SCDB microspheres, control experiments were designed to confirm the optimal primer 1-N addition concentration of microspheres of each particle size to reduce the primer waste in the preparation process of DNA-encoded capture primers. Calculations show that compared with the preparation of 96 DNA-encoded capture primers, the cost of preparing SCDB microspheres is only 15% of that. Compared with Smart-seq2, SCDB-seq uses Mg 2+ Heat shearing fragments mRNA molecules, eliminating the need for expensive Tn5 transposase to shear cDNA. Furthermore, thanks to the multiplexing nature of SCDB-seq, except for the mRNA reverse transcription process, each experimental step consumes only a single reagent. Therefore, when constructing transcriptome sequencing libraries for 96 samples using SCDB-seq, the reagent costs are approximately 13% of those required using Smart-seq2.

[0034] 3. This method can construct selective, highly multiplexed, medium-throughput (50-1000 samples) spatial transcriptome sequencing libraries for microregions on fresh-frozen (FF) and formalin-fixed and paraffin-embedded (FFPE) sample sections at single-cell resolution (~10 μm), thereby selectively obtaining gene expression profiles of multiple specific microregion samples in tissue sections. The experimental operation volume and reagent cost of this method are only ~1% and ~13% of those of existing similar methods, demonstrating good control of labor and reagent costs. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] Figure 1 Schematic diagram of the preparation of SCDB microspheres;

[0036] Figure 2 The diagram and results of quality control of complementary fluorescent probes are shown;

[0037] Figure 3 Schematic diagram for SCDB-seq sequencing library construction. DETAILED DESCRIPTION

[0038] Transfer 62.5 μL of 50 mg / mL 200 nm carboxylated magnetic beads to each of eight new 1.5 mL microcentrifuge tubes. Wash twice with 1 mL of 0.1 M MES buffer (pH 5.0). Dissolve the beads in 500 μL of 0.1 M MES buffer (pH 5.0). Add 62.5 μL of 50 mg / mL EDC solution and 50 μL of 100 μM primer 1-N to each 1.5 mL microcentrifuge tube, designating them "1-N." Incubate the eight 1.5 mL microcentrifuge tubes in a hybridization oven at 28°C and 1500 rpm for 5 hours. After magnetic separation, transfer the supernatant to another 1.5 mL microcentrifuge tube for Qubit assay and gel electrophoresis. Eight centrifuge tubes were washed three times with 1 ml of TET buffer (pH = 7.8), followed by magnetic separation. The microspheres were finally dissolved in 312.5 μl of 1× TET buffer (pH = 7.8) and stored at 4°C.

[0039] Resuspend the microspheres by pipetting, take 5μL of the microsphere suspension to a new 1.5ml centrifuge tube, and remove the supernatant by magnetic separation. Wash the microspheres twice by adding 100μL of QC solution, magnetically separating at room temperature, removing the supernatant, and adding QC solution to a final volume of 5μL. Add 1μL of 100μM fluorescent probe 1 to the 1.5ml centrifuge tube, pipette to mix, and then incubate the tube in the dark at room temperature for 30 minutes. Wash the microspheres three times with QC buffer to remove excess probe. The washing conditions are: 100μL QC buffer, vortex mixing, magnetic separation, and then remove the supernatant. At the end of the last wash, remove the supernatant, and the final volume in the centrifuge tube is 10μL. After vortexing the centrifuge tube, take 2.5μL three times onto a coverslip and observe under a fluorescence microscope.

[0040] After centrifugation, the eight 1.5ml centrifuge tubes were placed on a 1.5ml magnetic rack and the supernatant completely removed. The tubes were washed three times with 1x TE buffer. After the final wash, the supernatant was completely removed. Wash conditions were: 1 mL of 1x TE buffer followed by magnetic separation. To each of the eight 1.5ml centrifuge tubes, 32.5 μL of 10x isothermal amplification buffer, 235 μL of enzyme-free water, and 32.5 μL of 10 mM dNTPs were added. Mix to yield 300 μL of microsphere mix. In a new 96-well PCR plate, aliquot 1-N into wells 1-12 of rows A-H of the 96-well plate, 25 μL per well. Remove 100 μM primer 2-M from the refrigerator and centrifuge at 1000 g for 1 minute at room temperature. Primer 2-M was placed in a metal bath set at 72°C for 3 minutes, followed by 20 seconds at 4°C to remove primer dimers. Use a dispenser to add 6 μL of the corresponding primer 2-M (M = number of microwells) to the microwells of a 96-well PCR plate. Transfer the 96-well PCR plate to a thermostatic shaking metal bath and incubate at 85°C for 2 minutes. Carefully seal the tube with sealing film and store at -20°C. Prepare the isothermal reaction mix on ice according to Table 2.5 in a 1.5 ml centrifuge tube. Invert the tube 15 times to mix the mix and store on ice. Add 14 μL of the isothermal reaction mix to each well of the 96-well PCR plate. Then, return the 96-well PCR plate to the thermostatic shaking metal bath and incubate at 65°C for 120 minutes. Longer incubation times have no adverse effects.

[0041] Prepare 20 ml of newly prepared denaturing solution and add 50 μL of denaturing solution to each well of a 96-well PCR plate. Oscillate in a constant temperature oscillating metal bath at 85°C for 2 minutes. Centrifuge and remove the supernatant while hot. Repeat this step three times until the supernatant is completely removed. Wash the microspheres with neutralizing solution: Add 50 μL of neutralizing solution to each well of a 96-well PCR plate, vortex and centrifuge to remove the supernatant. Repeat this step three times. Add 50 μL of TET buffer to each well of a 96-well PCR plate. The SCDB microspheres are now prepared. According to the protocol "2.3.2 (2) Verifying the effect of primer fixation using fluorescent probes", use fluorescent probes 1 and 2 to verify whether the target oligonucleotide chain is present on the surface of the SCDB microspheres to determine whether the reaction is proceeding smoothly.

[0042] The amount of various solutions was calculated based on the number of pre-collected samples n. On an electronic thermostatic sample loading station precooled to 0°C, mRNA fragmentation mix (Fragmentation Mix-FF) was prepared: SMARTScribe first-strand reaction buffer, 5X, 2 μL; dNTP mix, (10 mM), 2 μL; MgCl2 (80 mM), 1 μL; Triton X-100, 0.5% (v / v), 1 μL; RNase inhibitor (40 U / ul), 1 μL; and template switching-reverse transcription mix (TS-RT Mix): Betame (5 M), 4 μL; DTT, 20 mM, 2 μL; dd H2O, 1 μL; 2S primer, 20 μM, 1 μL; SMARTScribe reverse transcriptase, 100 U / μL, 1 μL, each in n+n / 5 portions. Add 9.0 μL of Fragmentation Mix-FF and 1.0 μL of a 10 mg / mL SCDB microsphere suspension to a PCR tube or 96-well plate, respectively, and mix by vortexing. Collect the target tissue microregions into the cap of the PCR tube using laser-induced forward transfer (LIFT) or microregion sampling. Close the cap tightly and invert the PCR tube to allow the solution to soak into the target tissue, permeabilizing and releasing RNA molecules. Place the PCR tube or 96-well plate upside down in a thermostatic metal bath and perform lysis according to the following protocol: 60°C for 20 min. Briefly centrifuge the PCR tube to concentrate the solution at the bottom of the tube. Complete lysis of the target sample is monitored microscopically. If complete lysis is achieved, remove the PCR tube or 96-well plate and add 10 μL of LCM TS-RT Mix to each well. Vortex to mix, then place the PCR tube or 96-well plate in a thermostatic metal bath and perform reverse transcription according to the following protocol: 42°C for 30 min, 70°C for 10 min, and store at 4°C. The PCR tube or 96-well plate was then shaken, and the microsphere suspension was pipetted into a PCR tube. After magnetic adsorption, part of the supernatant was removed, and 20 μL was retained. Pre-amplification PCR Mix-FF was prepared as follows: HiFi HotStart ReadyMix, 2X, 25 μL; 20 μM 1S-Primer (PCR), 2.5 μL; 20 μM 2S-Primer (PCR), 2.5 μL. The PCR tube or 96-well plate was then placed in a PCR thermal cycler and pre-amplified at 98°C for 5 min; (98°C, 15 s; 60°C, 30 s; 72°C, 2 min) for 20 cycles; 72°C, 5 min; 4°C, ∞. The cDNA library was purified and concentrated using DNA purification magnetic beads.

[0043] Prepare the PCR amplification library construction Mix-FF: HiFi HotStart ReadyMix, 2X, 25 μL; 20 μM P7 indexed, 2.5 μL; 20 μM P5 universal, 2.5 μL. Add these to the above PCR tubes. Place the PCR tubes in a PCR thermocycler and perform PCR amplification to construct the sequencing library according to the following cycle: 98°C, 5 min; (98°C, 15 s; 60°C, 30 s; 72°C, 30 s) for 15 cycles; 72°C, 2 min; and 4°C, ∞. Purify and concentrate the cDNA library using DNA purification magnetic beads. Finally, perform Qubit assay on the cDNA and perform Aligent electrophoresis. If confirmed, send the library for analysis.

[0044] Figure 1 Schematic diagram of the SCDB microsphere preparation principle: (A) Schematic diagram of the principle of encoding microsphere synthesis based on equal division combination, showing the synthesis of 96 encoding microspheres through two rounds of equal division; (B) Bright field image of 20μm carboxyl magnetic microspheres; (C) Schematic diagram of the synthesis scheme and process of encoding microspheres based on equal division combination; (D) Primer design of primers 1-N and 2-M.

[0045] Figure 2 Schematic diagram and results of quality control of complementary fluorescent probes: (A) Schematic diagram of using fluorescent primers to characterize encoded microspheres; (B-D) Characterization results of fluorescent primer 1, fluorescent primer 2 and their fluorescence signal-to-noise ratio.

[0046] Figure 3 Schematic diagram of SCDB-seq sequencing library construction: (A) Schematic diagram of SCDB-seq experimental process; (B) Schematic diagram of SCDB-seq library preparation process.

Claims

1. A method for constructing a spatial transcriptome sequencing library based on equally distributed combinatorially encoded microspheres, characterized by: The steps include: (1) Design and preparation of SCDB microspheres: Two primers with specific sequence designs were used: primer 1-N, where 1≤N≤8, and primer 2-M, where 1≤M≤12. DNA barcode primers were prepared on the surface of the microspheres using an equal division combination preparation method. A certain amount of coded microspheres were divided into 8 parts and placed in centrifuge tubes and named 1-N. The corresponding primers 1-N were added to the centrifuge tubes for the first round of coding. Subsequently, the microspheres in the 8 centrifuge tubes were divided into a 96-well PCR plate in a specific order, i.e., centrifuge tubes 1-1 to 1-8 corresponded to rows A to H on the 96-well PCR plate, and the corresponding primers 2-M were added for the second round of coding. Finally, 96 coded microspheres with unique DNA barcode combinations were generated using 1-ethyl-(3 -dimethylaminopropyl) carbodiimide hydrochloride converts the carboxyl groups on the microsphere surface into O-acylisourea intermediates that can react with amino groups. This intermediate can quickly form an amide bond with primer 1-N modified with an amino group at the 5' end, covalently fixing primer 1-N to the microsphere surface. The 3' end of primer 2-M can produce reverse complementarity with the 3' end of primer 1-N. Subsequently, under the action of isothermal amplification DNA polymerase, the reverse complement of the sequence in primer 2-M is extended to the 3' end of primer 1-N, thereby completing the preparation of the microsphere surface encoding primer; (2) Design and use of SCDB-seq: SCDB microspheres are used to capture mRNA in samples, the captured mRNA molecules are reverse transcribed, a cDNA library that meets the requirements of second-generation sequencing is constructed through PCR amplification, and the target cDNA fragments are enriched using purification magnetic beads; (3) Design and implementation of spatial transcriptome sequencing based on SCDB-seq: The experimental protocol applied to fresh frozen tissue sections added 0.5% concentration of Triton X-100 to permeabilize the cell membrane during the mRNA capture process, allowing the mRNA to be fully released from the cells and captured by SCDB microspheres; the experimental protocol applied to FFPE tissue sections, in addition to adding 0.5% concentration of Triton X-100, eliminated the MgCl2 solution used to fragment the mRNA because the mRNA in the FFPE tissue sections had already been degraded to a certain extent. 2+ During the heat treatment step, proteinase K at a concentration of 0.125 μg / μL was added during the mRNA release and capture step and incubated at 60°C for 1 hour to fully remove the intermolecular cross-links in the FFPE tissue and release the mRNA molecules.

2. The method for constructing a spatial transcriptome sequencing library based on equally divided combinatorial encoding microspheres according to claim 1, characterized in that: The sequence design of primers 1-N, i.e., 5'→3', is: 1) PCR primer, used for amplifying cDNA library by PCR; 2) DNA bar 1-N, a specific barcode sequence that plays a DNA coding role, a total of 8 types; 3) a linker sequence, used for reverse complementation with the 3' end of primer 2-M; the sequence design of primer 2-M, i.e., 5'→3', is: 1) poly-dA, used for generating poly-dT with mRNA capture function; 2) UMI*, a sequence that plays a specific molecular recognition role; 3) DNA bar 2-M*, a specific coding sequence that plays a DNA coding role, a total of 12 types; 4) a reverse complementary linker sequence, used for reverse complementation with the 3' end of primer 1-N.

3. The method for constructing a spatial transcriptome sequencing library based on equally divided combinatorial encoding microspheres according to claim 1, characterized in that: The microspheres are monodisperse magnetic microspheres, the inner core of which is SiO2 or polystyrene, and the outer core is covered with a layer of Fe3O4 and the surface is coated with a chemical modification layer modified to carboxyl groups.

4. The method for constructing a spatial transcriptome sequencing library based on equally divided combinatorial encoding microspheres according to claim 1, characterized in that: The particle size of the microspheres is 0.1 μm to 100 μm.

Images