Multi-omics library construction method, detection method and related kit

Abstract

The invention discloses a multi-omics library construction method, a multi-omics library detection method and a related kit. The multi-omics library method comprises the following steps: placing a slice sample on a chip with a PolyA capturing function to carry out protein capturing and mRNA capturing, and carrying out protein capturing by using a nucleic acid coupled antibody; respectively carrying out reverse transcription and complementary chain synthesis on the captured mRNA and the nucleic acid sequence on the nucleic acid coupling antibody to respectively obtain cDNA and the complementary chain of the nucleic acid sequence; and performing library construction on complementary chains of the cDNA and the nucleic acid sequence to obtain a transcriptome library and a proteome library of the slice sample. According to the present invention, by using the PolyA and the antibody coupling nucleic acid, the simultaneous capture of the antibody coupling nucleic acid PolyA and the mRNA combined with the antigen protein is achieved by using the PolyT structure on the chip, and the simultaneous construction and the subsequent in-situ detection of the proteome library and the transcriptome library are achieved so as to achieve the whole tissue visual field detection of the transcriptome and the proteome.

Description

Mult-omics Library Construction Method, Detection Method and Related Kits

[0001] The present invention relates to the technical field of multi-omics detection. Specifically, it relates to a mult-omics library construction method, a detection method and related kits.

[0002] Common techniques for detecting proteins include immunofluorescence and immunohistochemistry. Immunofluorescence is the gold standard for protein detection, but limited by fluorescence channels and antibody species, usually only the expression of 1-5 proteins can be detected on a single tissue section. Mature products on the market that can detect the expression of multiple proteins cannot simultaneously detect the expression of the transcriptome. For example, the products of Olink Proteomics can only detect the protein expression in liquid phase (blood, body fluid) and cannot detect the protein localization and expression in tissue sections. Although the products of Akoya Biosciences can detect the protein expression on tissue sections, they cannot detect the expression of the transcriptome. The GeoMx Digital Spatial Profiler technology of NanoString can detect both the transcriptome and the protein expression, but the detection field of view of this product is limited. When selecting regions of interest for a tissue, usually only dozens of regions with a size of 50-100 μm in diameter can be detected, and it is impossible to perform a full-field detection on a section of tissue. Although the Spatial Gene Expression product of 10×Genomics can simultaneously detect the expression of the whole transcriptome and proteome in a field of view of 0.65 cm × 0.65 cm, its resolution is too low, and a Spot is about 55 μm, which cannot reach the detection at the single-cell level.

[0003] Therefore, in terms of simultaneously detecting the proteome and the transcriptome, the existing technologies still need to be improved.

[0004]

[0005] The main object of the present invention is to provide a mult-omics library construction method, a detection method and related kits to provide a new solution for detecting the transcriptome and proteome with a full-tissue field of view.

[0006] To achieve the above object, according to one aspect of the present invention, a method for constructing a multi-omics library is provided. The method includes: Step S1, placing a sliced sample on a chip with the function of capturing PolyA for protein capture and mRNA capture. Among them, nucleic acid-conjugated antibodies are used for protein capture. The nucleic acid-conjugated antibodies include antibodies and nucleic acid sequences conjugated to the antibodies. The nucleic acid sequences sequentially include, in the direction away from the antibody: a PCR adapter region, an antibody barcode region, and a Poly A sequence. The chip has Poly T. The nucleic acid-conjugated antibodies bind to Poly T on the chip through the Poly A sequence to capture the antigen proteins bound by the antibodies. The mRNA in the sliced sample binds to Poly T on the chip through Poly A for mRNA capture; Step S2, respectively performing reverse transcription and synthesis of complementary strands on the captured mRNA and the nucleic acid sequences on the nucleic acid-conjugated antibodies bound to antigens, and respectively obtaining cDNA and complementary strands of the nucleic acid sequences on the nucleic acid-conjugated antibodies; Step S3, constructing libraries for the cDNA and the complementary strands of the nucleic acid sequences of the nucleic acid-conjugated antibodies to obtain a transcriptome library and a proteome library of the sliced sample.

[0007] Further, Step S1 includes: Step S11, sealing the chip with a sealing solution to obtain a sealed chip; Step S12, incubating the nucleic acid-conjugated antibodies with the sealed chip to obtain a protein capture chip; Step S13, performing a permeabilization treatment on the protein capture chip to achieve the capture of mRNA and nucleic acid sequences, and obtaining a permeabilization-treated chip.

[0008] Further, Step S2 includes: respectively performing reverse transcription and synthesis of complementary strands on the captured mRNA and nucleic acid sequences on the permeabilization-treated chip to obtain synthesis products. The synthesis products include eDNA and complementary strands of nucleic acid sequences; preferably, the temperature of reverse transcription is 40°C to 44°C, and the time is 1.5 hours to 3 hours.

[0009] Further, Step S3 includes: Step S31, sequentially performing tissue removal and probe digestion on the sliced sample after reverse transcription and synthesis of complementary strands on the permeabilization-treated chip to obtain a digestion solution containing synthesis products; Step S32, separating and purifying the synthesis products from the digestion solution, and respectively amplifying to obtain a proteome library and a transcriptome library.

[0010] Furthermore, the blocking solution comprises: 3×SSC, 5% - 10% (v / v) serum, 0.1% (v / v) Triton-x-100, 0.1 mg / mL - 1 mg / mL salmon sperm DNA, 0.5 μM - 10 μM of 22bp - 32bp PolyA, and 5% (v / v) RNase inhibitor; preferably, the concentration of 22bp - 32bp PolyA in the blocking solution is 1 μM - 10 μM, more preferably 5 μM - 10 μM, and even more preferably 8 μM - 10 μM; preferably, the concentration of salmon sperm DNA in the blocking solution is 0.5 mg / mL - 1 mg / mL.

[0011] Furthermore, in step S12, before incubating the nucleic acid-conjugated antibody with the blocked chip, it further includes a step of diluting the nucleic acid-conjugated antibody with an antibody diluent, wherein the antibody diluent is selected from any one of the following: the blocking solution, a PBS buffer containing PolyA and DNA, or a 2 - 3×SSC buffer containing PolyA and DNA. In the PBS buffer and the SSC buffer, the concentration of PolyA is 0.5 μM - 10 μM, and the concentration of DNA is 0.1 mg / ml - 1 mg / ml. Preferably, the DNA is salmon sperm DNA.

[0012] Furthermore, step S12 includes: incubating the nucleic acid-conjugated antibody with the blocked chip so that the nucleic acid-conjugated antibody binds to the antigen to obtain an antibody-antigen complex; placing the antibody-antigen complex in 0.1×SSC buffer at 60°C - 70°C, preferably 65°C - 70°C, and reacting for 5 - 15 min, preferably 10 min, to obtain a protein capture chip.

[0013] Furthermore, the temperature of the permeabilization treatment is 35°C - 38°C, and the time of the permeabilization treatment is 3 min - 20 min.

[0014] Furthermore, the temperature of tissue removal is 50°C - 60°C, and the time of tissue removal is 20 - 40 min; preferably, the temperature of digestion is 50°C - 60°C, and the time of digestion is 2.5 hours - 4 hours.

[0015] Furthermore, the Poly T on the chip is multi-clustered, and the distance between adjacent two clusters is 495 nm to 505 nm; preferably, in the nucleic acid-conjugated antibody, the length of the PCR linker region is 18 bp to 23 bp, the length of the antibody barcode region is 8 bp to 20 bp, and the length of PolyA is 22 bp to 35 bp; preferably, the antibody part in the nucleic acid-conjugated antibody is selected from any one or more of the following antibodies: CD169 antibody, CD3 antibody, CD8 antibody, CD19 antibody, CD20, CD21, CD45R-B220, CD163, CD38, CD11b, CD140a, CD335, CD371, CD90.2, TER-119, CD274CD279, CD56, CD14, CD340, CD324, CD38, CD29, CD68, CD44, CD21 / 35, IgD, CD5, CD4, IgM, CD79b, CD11c, F4 / 80, CD27, CD31 and CD8a.

[0016] According to the second aspect of the present application, there is provided a multi-omics detection method, which includes: constructing a proteome library and a transcriptome library of a sample section to be tested by using any one of the above multi-omics library construction methods; sequencing the proteome library and the transcriptome library to obtain the proteome and transcriptome information of the sample section.

[0017] According to the third aspect of the present application, there is provided a multi-omics library construction kit, which includes any of the following: PolyA capture chip, nucleic acid-conjugated antibody, blocking solution, tissue permeabilization solution, tissue removal solution and digestion solution; wherein, the blocking solution includes: 3×SSC, 5% to 10% (v / v) serum, 0.1% (v / v) Triton-x-100, 0.1 mg / mL to 1 mg / mL salmon sperm DNA, 0.5 μM to 10 μM of 22 bp to 32 bp PolyA and 5% (v / v) ribonuclease inhibitor.

[0018] Furthermore, the concentration of 22bp - 32bp PolyA in the blocking solution is 1 μM - 10 μM, more preferably 5 μM - 10 μM, and even more preferably 8 μM - 10 μM; preferably, the concentration of salmon sperm DNA in the blocking solution is 0.5 mg / mL - 1 mg / mL; preferably, the Poly A capture chip refers to a capture chip containing Poly T, and the nucleic acid-conjugated antibody includes an antibody and a nucleic acid sequence conjugated to the antibody. The nucleic acid sequence sequentially includes, in the direction away from the antibody: a PCR linker region, an antibody barcode region, and a Poly A sequence; preferably, the PolyT on the PolyA capture chip is in multiple clusters, and the distance between adjacent two clusters is 495 nm - 505 nm; preferably, in the nucleic acid-conjugated antibody, the length of the PCR linker region is 18 - 23 bp, the length of the antibody barcode region is 8 - 20 bp, and the length of PolyA is 22 bp - 35 bp.

[0019] Applying the technical solution of the present invention, by using an antibody-conjugated nucleic acid with a PolyA and an antibody recognition sequence library amplification sequence, and utilizing the PolyT structure on the chip, it is possible to simultaneously capture the antibody-conjugated nucleic acid PolyA bound to the antigen protein and the transcriptome PolyA, thereby realizing the simultaneous construction of the proteome library and the transcriptome library, as well as the subsequent co-detection of the proteome and the transcriptome.

[0020] The accompanying drawings of the specification, which form a part of this application, are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation to the present invention. In the drawings:

[0021] Figure 1 shows a schematic structural diagram of the nucleic acid-conjugated antibody used in the preferred embodiment of the present application;

[0022] Figure 2 shows a schematic diagram of the principle of multi-omics library construction in the preferred embodiment of the present application;

[0023] Figure 3 shows a multi-omics detection result diagram in the first embodiment of the present application;

[0024] Figure 4 shows a multi-omics detection result diagram in the second embodiment of the present application;

[0025] Figure 5 shows a multi-omics detection result diagram in the third embodiment of the present application;

[0026] Figure 6 shows a multi-omics detection result diagram in the fourth embodiment of the present application.

[0027] Figure 7 shows a multi-omics detection result diagram in the fifth embodiment of the present application.

[0028] Figure 8 shows a multi-omics detection result diagram in the sixth embodiment of the present application.

[0029] Figure 9 shows the multi-omics detection result diagram in the seventh embodiment of the present application.

[0030] Figure 10 shows the multi-omics detection result diagram in the eighth embodiment of the present application.

[0031] Figure 11 shows the multi-omics detection result diagram in the ninth embodiment of the present application.

[0032] Figure 12 shows the multi-omics detection result diagram in the tenth embodiment of the present application.

[0033] Figure 13 shows the multi-omics detection result diagram in the eleventh embodiment of the present application.

[0034] Figure 14 shows the multi-omics detection result diagram in the twelfth embodiment of the present application.

[0035] It should be noted that, without conflict, the embodiments in the present application and the features in the embodiments may be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0036] As mentioned in the background art, there is still room for improvement in the prior art in terms of the solution that can detect both the transcriptome and the proteome in the whole tissue view. To improve this situation, the inventors of the present application tried to use the method of antibody-conjugated nucleic acid based on the self-developed spatial and temporal omics technology, and utilize the PolyT structure on the chip to achieve the simultaneous capture of antibody-conjugated nucleic acid PolyA bound to the antigen protein and transcriptome PolyA, thereby realizing the simultaneous construction of the proteome library and the transcriptome library, and the co-location detection of the proteome and the transcriptome. On this basis, the applicant proposed a series of protection solutions for the present application.

[0037] In a typical embodiment of the present application, a method for constructing a multi-omics library is provided. The method includes: Step S1, placing a section sample on a chip with the function of capturing PolyA for protein capture and mRNA capture. Among them, nucleic acid-conjugated antibodies are used for protein capture. As shown in Figure 1, the nucleic acid-conjugated antibodies include antibodies and nucleic acid sequences conjugated to the antibodies. The nucleic acid sequences sequentially include, in the direction away from the antibody: a PCR linker region (for example, it can be 21bp), an antibody barcode region (for example, it can be 15bp), and a PolyA (for example, it can be 32bp) sequence. The chip has PolyT, and the nucleic acid-conjugated antibodies bind to PolyT on the chip through the PolyA (32bp) sequence to capture the antigen proteins bound by the antibodies, and the mRNA in the section sample is captured by binding PolyA to PolyT on the chip; Step S2, respectively performing reverse transcription and synthesis of complementary strands on the nucleic acid sequences on the captured mRNA and the nucleic acid-conjugated antibodies bound to proteins to obtain cDNA sequences and complementary strands of the nucleic acid sequences of the nucleic acid-conjugated antibodies; Step S3, constructing libraries for the cDNA sequences and the complementary strands of the nucleic acid sequences of the nucleic acid-conjugated antibodies to obtain a proteome library and a transcriptome library of the section sample.

[0038] In the present application, spatiotemporal proteomics is achieved by using a class of nucleic acid-conjugated antibodies (commercial products can be used, for example, the Totalseq-A series of antibodies sold by Biolegend). This class of antibodies is conjugated with a nucleic acid sequence on the basis of normal antibodies. This nucleic acid sequence consists of three parts, namely, a PCR linker region (used for subsequent amplification of the library, and the specific length varies according to different antibodies, for example, it can be 21bp), an antibody barcode region (used to identify different antibodies, and the specific length can also be reasonably set, for example, it can be 15bp), and a PolyA (used to bind to Poly T on the chip and be fixed at a specific position on the chip, and the specific length can also be appropriately adjusted, for example, it can be 32bp) sequence.

[0039] The method for constructing a multi-omics library in the present application uses an antibody-conjugated nucleic acid with PolyA and an antibody recognition sequence library amplification sequence, and utilizes the PolyT structure on the chip to simultaneously capture the antibody-conjugated nucleic acid PolyA and the transcriptome PolyA, thereby simultaneously constructing a proteome library and a transcriptome library, and subsequent co-detection of the proteome and the transcriptome, so as to achieve the detection of the transcriptome and the proteome in the whole tissue view.

[0040] In a preferred embodiment, the above step S1 includes: step S11, blocking the chip with a blocking solution (to reduce the occurrence of non-specific hybridization) to obtain a blocked chip; step S12, incubating the nucleic acid-conjugated antibody with the blocked chip to obtain a protein capture chip; step S13, performing a permeabilization treatment on the protein capture chip to achieve the capture of mRNA, thereby obtaining a permeabilization-treated chip.

[0041] As described above, in the nucleic acid sequence of the nucleic acid-conjugated antibody, the PCR linker region is used for subsequent library amplification, the antibody barcode region is used to interpret the antibody type, and the barcode sequences of the same antibody are the same. The PolyA sequence can hybridize with the PolyT of the probe on the chip. The antibody part of this nucleic acid-conjugated antibody still has biological activity, so it can bind to the corresponding antigen epitope on the tissue section sample. In the above preferred embodiment, in order to minimize the hybridization of the conjugated nucleic acid with the probe on the chip, thereby triggering non-specific signals, the chip is blocked with a blocking solution before antibody incubation.

[0042] In a preferred embodiment, the blocking solution includes: 3×SSC at a final concentration, 5% - 10% (v / v) serum (such as 5%, 6%, 7%, 8%, 9% or 10%), 0.1% (v / v) Triton-x-100, 0.1 mg / mL - 1 mg / mL salmon sperm DNA (such as 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, 0.4 mg / mL, 0.5 mg / mL, 0.6 mg / mL, 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL, 1 mg / mL), 0.5 μM - 10 μM of 22 bp - 32 bp PolyA (such as 0.5 μM, 0.6 μM, 0.7 μM, 0.8 μM, 0.9 μM, 1.0 μM, 1.5 μM, 2.0 μM, 2.5 μM, 3.0 μM, 3.5 μM, 4.0 μM, 4.5 μM, 5.0 μM, 5.5 μM, 6.0 μM, 6.5 μM, 7.0 μM, 7.5 μM, 8.0 μM, 8.5 μM, 9.0 μM, 9.5 μM, 10.0 μM) and 5% (v / v) ribonuclease inhibitor. Preferably, the concentration of 22 bp - 32 bp PolyA in the blocking solution is 1 μM - 10 μM, more preferably 5 μM - 10 μM, and further preferably 8 μM - 10 μM; preferably, the concentration of salmon sperm DNA in the blocking solution is 0.5 mg / mL - 1 mg / mL.

[0043] In the above preferred embodiment, the pH value of the blocking solution is preferably 6 - 9. In the above preferred embodiment, the salt ion concentration of the blocking solution is applicable between 1× and 7×, and there is no obvious adverse effect on transcriptome capture.

[0044] In this application, the inventors further found through experiments that: 1) When the blocking solution contains 10 μM of 22-32 bp PolyA, the blocking effect is better than that of 0.5 μM of 22-32 bp PolyA. 2) When the blocking solution contains 0.1 mg / ml - 1 mg / ml of DNA (such as salmon sperm DNA), the effect is better than that without adding DNA.

[0045] The inventors also found that: 3) Using the blocking solution directly or a PBS buffer or 2 - 3×SSC buffer containing 0.5 μM - 10 μM PolyA (22-32 bp) and 0.1 - 1 mg / ml of DNA (salmon sperm DNA) as the antibody diluent is better than using PBS or 2 - 3×SSC directly. 4) When the blocking solution and the antibody diluent do not contain PolyA (22-32 bp), it is extremely easy to cause experimental failure and too high background. 5) When the antibody diluent does not contain PolyA (22-32 bp), it is easy to cause unstable results and the background is higher than the signal.

[0046] Therefore, in a preferred embodiment, in step S12, before incubating the nucleic acid-conjugated antibody with the blocked chip, it further includes the step of diluting the nucleic acid-conjugated antibody with an antibody diluent, where the antibody diluent is selected from any one of the following: the blocking solution, a PBS buffer containing PolyA and DNA, or a 2 - 3×SSC buffer containing PolyA and DNA. In the PBS buffer and the SSC buffer, the concentration of PolyA is 0.5 μM - 10 μM, and the concentration of DNA is 0.1 mg / ml - 1 mg / ml. Preferably, the DNA is salmon sperm DNA.

[0047] In a preferred embodiment, in step S12, after incubating the nucleic acid-conjugated antibody with the blocked chip so that the nucleic acid-conjugated antibody binds to the antigen, an antibody-antigen complex is obtained; the antibody-antigen complex is placed at 60°C - 70°C (such as 60°C, 61°C, 62°C, 63°C, 64°C, 65°C, 66°C, 67°C, 68°C, 69°C or 70°C), preferably at 65°C - 70°C, and reacted in 0.1×SSC buffer for 5 - 15 min to remove the non-specific binding of the nucleic acid-conjugated antibody to the tissue or the chip, preferably for 10 min, to obtain a protein capture chip.

[0048] In the above preferred embodiments, to minimize the hybridization of the coupled nucleic acids with the probes on the chip, thereby avoiding non-specific signals, after optimizing the blocking solution or antibody-antigen binding, a reaction is carried out at 60 °C to 70 °C in 0.1×SSC buffer (it should be noted that the 0.1×SSC buffer here is not the only limiting and feasible one, but exemplary. For example, 3×SSC buffer also has a similar effect). A 10-minute reaction can detect protein expression without affecting the capture level of the transcriptome. Among them, the optimization of the blocking solution formulation is mainly reflected in using salmon sperm DNA to block the sites on the tissue that adsorb nucleic acids, and by using PolyA in the blocking solution to block the non-specific signals of the hybridization between the probes outside the tissue and the PolyA conjugated to the antibody. And by adopting the method of high temperature and low salt, the non-specific signals of the hybridization between the probes and the PolyA conjugated to the antibody caused by insufficient blocking can be removed. Then, through the specific binding reaction of antibody-antigen, the nucleic acid sequence part conjugated to the antibody hybridizes with the PolyT of the probe near the antigen. At the same time, the mRNA in the tissue can also hybridize with the PolyT of the probe.

[0049] In a preferred embodiment, step S2 includes: respectively performing reverse transcription and complementary strand synthesis on the mRNA captured on the permeabilized chip and the nucleic acid sequence on the nucleic acid-conjugated antibody bound to the captured antigen protein to obtain synthesis products, where the synthesis products include cDNA and the complementary strand of the nucleic acid sequence of the nucleic acid-conjugated antibody; preferably, the temperature of reverse transcription is 40 °C to 44 °C, and the time is 1.5 hours to 3 hours.

[0050] In a preferred embodiment, step S3 includes: step S31, sequentially performing tissue removal and probe digestion on the section sample on the permeabilized chip after reverse transcription and the complementary strand synthesis to obtain a digestion solution containing the above synthesis products; step S32, separating and purifying the above synthesis products from the digestion solution, and respectively amplifying to obtain a proteome library and a transcriptome library.

[0051] As shown in Figure 2, after the reverse transcription reaction, the PolyT probe extends the barcode and PCR adapter sequence of the antibody, and at the same time, the PolyT probe hybridized with mRNA can also extend the cDNA sequence. In this way, the PolyT probe contains the information of the antibody type, mRNA information, and the position information on the chip. In addition, since the transcriptome library fragments are about 1.5 kb, and the protein library fragments are about 165 bp, different amounts of magnetic beads are used to separate the libraries of different fragments. For example, 0.8× magnetic beads are used to enrich and purify the transcriptome, and 2× magnetic beads are used to purify the proteome. By using the method of differential enrichment and purification with magnetic beads, the transcriptome and proteome libraries can be separated.

[0052] The tissue permeabilization solution used in the above tissue permeabilization treatment step can be an existing permeabilization solution that can achieve the permeabilization effect of tissue sections. In a preferred embodiment, tissue permeabilization is performed using a tissue permeabilization solution. Preferably, the temperature of the permeabilization treatment is 35-38 °C, and the time of the permeabilization treatment is 3-20 min.

[0053] In a preferred embodiment, tissue removal is performed using a tissue removal solution, and digestion is performed using a digestion solution. Preferably, the temperature of tissue removal is 50 °C to 60 °C, and the time of tissue removal is 20-40 min; preferably, the temperature of digestion is 50 °C to 60 °C, and the time of digestion is 2.5-4 hours.

[0054] It should be noted that the above tissue removal solution, digestion solution, and permeabilization solution can all use the corresponding products in existing commercially available kits. For example, in the Spatial Transcriptome Kit of BGI: STOmies Gene Expression Reagent Set - S1 (Catalog No.: 1000028496), the tissue removal solution is TR buffer, the digestion solution is eDNA release buffer, and the permeabilization solution is PR Enzyme.

[0055] In a preferred embodiment, the Poly T on the chip is multiple clusters, and the distance between adjacent two clusters is 495-505 nm; preferably, in the nucleic acid-conjugated antibody, the length of the PCR adapter region is 18-23 bp, the length of the antibody barcode region is 8-20 bp, and the length of PolyA is 22-35 bp. It should be noted that the nucleic acid-conjugated antibody in this application is not particularly limited and can be reasonably selected according to the research purpose. For example, it can be selected from any one or more of the following antibodies: CD169 antibody, CD3 antibody, CD8 antibody, CD19 antibody, CD20, CD21, CD45R-B220, CD163, CD38, CD11b, CD140a, CD335, CD371, CD90.2, TER-119, CD274 CD279, CD56, CD14, CD340, CD324, CD38, CD29, CD68, CD44, CD21 / 35, IgD, CD5, CD4, IgM, CD79b, CD11c, F4 / 80, CD27, CD31, and CD8a.

[0056] In the above preferred embodiment, the distance between two Poly T cluster probes on the chip is set at about 500 nm, which is convenient for achieving co-location detection of the whole transcriptome and proteome at a resolution of 0.5 μm. Since the diameter of a single cell is usually about 10 μm, the chip of this application can achieve multi-omics detection at single-cell resolution, and the protein detection effect is close to that of immunofluorescence (the gold standard, resolution 0.2 μm).

[0057] In the second typical embodiment of the present application, a multi-omics detection method is provided, which includes: constructing a proteome library and a transcriptome library of a sample section to be tested by using any of the foregoing multi-omics library construction methods; sequencing the proteome library and the transcriptome library to obtain proteome and transcriptome information of the section sample.

[0058] By using the multi-omics detection method of the present application, not only can co-location detection of the proteome and the transcriptome be achieved, but also full-field detection at the single-cell scale can be achieved. In addition, 30 or more proteins can be detected simultaneously.

[0059] In the third typical embodiment of the present application, a multi-omics library construction kit is provided, which includes any of the following: Poly A capture chip, nucleic acid-conjugated antibody, blocking solution, tissue permeabilization solution, tissue removal solution, and digestion solution, and necessarily includes a blocking solution. Among them, the blocking solution includes: 3×SSC, 5% - 10% (v / v) serum, 0.1% (v / v) Triton-x-100, 0.1 mg / mL - 1 mg / mL salmon sperm DNA, 0.5 μM - 10 μM 22bp - 32bp PolyA, and 5% (v / v) RNase inhibitor.

[0060] In some preferred embodiments, the concentration of 22bp - 32bp PolyA in the above blocking solution is 1 μM - 10 μM, more preferably 5 μM - 10 μM, and further preferably 8 μM - 10 μM; preferably, the concentration of salmon sperm DNA in the blocking solution is 0.5 mg / mL - 1 mg / mL; preferably, the Poly A capture chip refers to a capture chip containing Poly T, and the nucleic acid-conjugated antibody includes an antibody and a nucleic acid sequence conjugated to the antibody. The nucleic acid sequence includes, in the direction away from the antibody, a PCR adapter region, an antibody barcode region, and a Poly A sequence in sequence; preferably, the Poly T on the Poly A capture chip is in multiple clusters, and the distance between adjacent clusters is 495 nm - 505 nm; preferably, in the nucleic acid-conjugated antibody, the length of the PCR adapter region is 18 - 23bp, the length of the antibody barcode region is 8 - 20bp, and the length of PolyA is 22bp - 35bp.

[0061] In a preferred embodiment, the multi-omics library construction method, detection method, and kit formulation are as follows:

[0062] (1) Frozen tissue section (10 μm), after the tissue is dried by baking, fix it with -20°C pre-cooled methanol for 30 min; or fix it with 4% PFA at room temperature for 10 min.

[0063] (2) Add 100 μl of ssDNA for staining (a nucleic acid dye for staining cell nuclei, or DAPI or other dyes can also be used for staining), react at room temperature for 5 min, wash away the liquid, and wash 3 times with 0.1×SSC.

[0064] (3) Mount the slide with 5 μl of glycerol, take fluorescence images by photographing, and soak the 24-well plate in 500 μl of 5×SSC for 5 min.

[0065] Note: The above steps (2) and (3) are only for staining cell nuclei, and staining cell nuclei is not an essential step for library construction.

[0066] (4) Take out the chip, add 50 μl of washing buffer for washing, react for 1 min, wash away the liquid, repeat this step, and wash 3 times in total with the washing buffer.

[0067] (5) Add 50 μl of blocking solution, react at room temperature for 30 min. Poly A in the blocking solution can block the probes outside the tissue area on the chip (removing hybridization background), and serum and salmon sperm DNA can block the sites on the tissue that non-specifically adsorb conjugated antibodies.

[0068] (6) Add the pre-prepared antibody, react at room temperature for 45 min to allow the antibody to fully bind to the antigen.

[0069] (7) Wash away the antibody, wash with 0.1×SSC, react for 1 min, repeat the washing 5 times, and air-dry the tissue.

[0070] (8) React at 70 °C with 0.1×SSC for 10 min, and then wash 3 times with 0.1×SSC.

[0071] (9) Permeabilize the tissue with the tissue permeabilization solution for an appropriate time, usually 12 min at 37 °C. Then add RT mix and react at 42 °C for 2 hours.

[0072] (10) Add 400 μl of tissue removal solution, digest at 55 °C for 30 min, remove the tissue removal solution, add 400 μl of digestion solution, and digest at 55 °C for 3 hours.

[0073] (11) Purify cDNA from the digestion solution, amplify the protein library and transcriptome library respectively, prepare DNA nanoballs (DNBs), and load them onto the machine for sequencing.

[0074] For the transcriptome library and protein library, use the DNB library preparation method of BGI sequencing platform and sequence with MGI2000. For the protein library, sequence 50 bp for the first strand and 15 bp for the second strand; for the transcriptome library, sequence 50 bp for the first strand and 50 bp for the second strand. Perform data analysis through the bioinformatics analysis process to obtain specific information and restore the transcriptome and proteome to specific positions on the chip.

[0075] The beneficial effects of the present application will be further illustrated below in conjunction with specific embodiments.

[0076] Example 1

[0077] I. Experiment organization: 10-μm frozen sections of mouse spleen tissue.

[0078] II. Reagent formulations:

[0079] 1) Blocking solution:

[0080] Table 1:

[0081] 2) Antibody diluent: The same as the blocking solution.

[0082] 3) Washing buffer (WB):

[0083] Table 2:

[0084] 4) Primary antibody incubation solution: Dilute the antibody with the antibody diluent. The antibody is CD169 (Biolegend, catalog number: 142425), and the dosage is 0.5 μl / 50 μl reaction system, with a final concentration of 0.25 μg / μl.

[0085] 5) Secondary antibody incubation solution: 0.1 μl Goat anti-Rat 555 / 50 μl reaction system, with a final concentration of 0.4 μg / μl.

[0086] III. Experimental steps:

[0087] 1) Take mouse spleen tissue, freeze tissue sections (10 μm), and fix with pre-cooled methanol (-20 °C) for 30 min.

[0088] 2) Take out the chip. After the methanol has evaporated and the tissue has become dry, add the washing buffer, 50 μl per chip, and react for 1 min.

[0089] 3) Aspirate the liquid and repeat step 2 twice, for a total of 3 washes.

[0090] 4) Add 50 μl of the blocking solution per chip and react at room temperature for 30 min.

[0091] 5) Aspirate the liquid and add 50 μl of the primary antibody incubation solution, and react at room temperature for 45 min.

[0092] 6) Aspirate the reaction solution, add 50 μl of the washing solution, react for 1 min, and repeat this step for a total of 3 washes.

[0093] 7) Add the secondary antibody incubation solution and react at room temperature for 30 min.

[0094] 8) Aspirate the reaction solution, add 50 μl of washing solution, and react at room temperature for 3 min.

[0095] 9) Aspirate the reaction solution, add 50 μl of washing solution, react for 1 min, repeat this step for a total of 3 washes, air-dry the chip. After the tissue is dry, add 3 μl of glycerol to cover the slide, and collect the fluorescence image.

[0096] Note: The above steps 1-9 are the experimental steps of immunofluorescence. The following are the protein capture steps:

[0097] 10) Remove the coverslip, place the chip in 5×SSC solution and react for 5 min to wash away the glycerol.

[0098] 11) Place the chip in a 24-well plate, add 1 ml of 0.1×SSC, and incubate at 65 °C for 10 min.

[0099] 12) Take out the chip, wash the chip once with 0.1×SSC, add the permeabilization reagent, and react at 37 °C for 12 min.

[0100] 13) Wash the chip once with 0.1×SSC, add RT mix, and react at 42 °C for 2 hours.

[0101] 14) Add 400 μl of tissue removal solution, digest at 55 °C for 30 min, remove the tissue removal solution, add 400 μl of digestion solution, and incubate at 55 °C for 3 hours.

[0102] 15) Purify cDNA from the digestion solution, amplify the protein library and transcriptome library respectively, prepare DNB, and load it onto the machine for sequencing.

[0103] 16) Among them, the transcriptome sequencing adopts a sequencing scheme of 50 bp for the first strand + 50 bp for the second strand, and the proteome sequencing adopts a sequencing scheme of 50 bp for the first strand + 15 bp for the second strand.

[0104] 17) Visualize the sequencing results: Data analysis can be carried out through the existing bioinformatics analysis process to obtain specific information and restore the transcriptome and proteome to specific positions on the chip.

[0105] IV. Experimental results and analysis:

[0106] As shown in Figure 3, the visualization result of CD169 has a high similarity with the corresponding IF (immunofluorescence). It can be seen from the locally enlarged figure on the right that the visualization is almost the same and can achieve the effect of immunofluorescence.

[0107] Example 2

[0108] I. Experimental tissue: 10-μm frozen sections of human tonsil tissue.

[0109] II. Reagent Formulations:

[0110] 1) Blocking Solution: Same as Table 1 in Example 1.

[0111] 2) Antibody Diluent: Same as the blocking solution.

[0112] 3) Washing Buffer (WB): Same as Table 2 in Example 1

[0113] 4) Primary Antibody Incubation Solution: Dilute the antibody with the antibody diluent. The dosage is 0.5 μl / 50 μl reaction system, and the final concentration is 0.25 μg / μl. Among them, the antibodies used are CD3 (Biolegend, catalog number: 300475); CD8 (Biolegend, catalog number: 344751); CD19 (Biolegend, catalog number: 302259).

[0114] 5) Secondary Antibody Incubation Solution: 0.1 μl Goat anti-Rat 555 / 50 μl reaction system, and the final concentration is 0.4 μg / μl.

[0115] III. Experimental Procedures:

[0116] 1) Take human tonsil tissue, prepare frozen tissue sections (10 μm), and fix with pre-cooled methanol (-20 °C) for 30 min;

[0117] 2) Add 100 μl of DAPI for nuclear staining, react at room temperature for 5 min, aspirate the liquid, and wash 3 times with 0.1×SSC.

[0118] 3) Mount with 5 μl of glycerol, take pictures to collect fluorescence images, and soak the 24-well plate in 500 μl of 5×SSC for 5 min.

[0119] 4) Take out the chip, add 50 μl of WB washing solution, react for 1 min, aspirate the liquid, repeat this step, and wash a total of 3 times with the WB washing solution.

[0120] 5) Add 50 μl of blocking solution and react at room temperature for 30 min.

[0121] 6) Add the pre-prepared primary antibody incubation solution and react at room temperature for 45 min to allow the antibody to fully bind to the antigen.

[0122] 7) Aspirate the antibody, wash with 0.1×SSC, react for 1 min, repeat washing 5 times, and air-dry the tissue.

[0123] 8) React at 70 °C with 3×SSC for 10 min, and then wash 3 times with 0.1×SSC.

[0124] 9) Permeabilize with tissue permeabilization solution for an appropriate time, usually 12 min at 37 °C is sufficient. Then add RT mix and react at 42 °C for 2 hours.

[0125] 10) Add 400 μl of tissue removal solution, digest at 55 °C for 30 min, remove the tissue removal solution, add 400 μl of digestion solution, and incubate at 55 °C for 3 hours.

[0126] 11) Purify cDNA from the digestion solution, amplify the protein library and transcriptome library respectively, prepare DNA nanoballs (DNBs), and load them onto the sequencer for sequencing.

[0127] 12) For the transcriptome library and protein library, use the DNB library preparation method of BGI sequencing platform and sequence with MGI2000. For the protein library, the first strand is sequenced for 50 bp and the second strand is sequenced for 15 bp; for the transcriptome library, the first strand is sequenced for 50 bp and the second strand is sequenced for 50 bp. Data analysis is performed through the bioinformatics analysis pipeline to obtain specific information and restore the transcriptome and proteome to specific positions on the chip.

[0128] IV. Results and Analysis:

[0129] As shown in Figure 4, the protein expression of CD3 / 8 / 19 and the transcriptome expression have similar expression patterns, and co - localization detection of the transcriptome and proteome is achieved on the same section.

[0130] Example 3

[0131] I. Experimental Tissue: Mouse spleen tissue.

[0132] II. Reagent Formulations:

[0133] 1) Blocking solution: The same as Table 1 in Example 1.

[0134] 2) Antibody dilution solution: The same as the blocking solution.

[0135] 3) Wash buffer (WB): The same as Table 2 in Example 1.

[0136] 4) Primary antibody incubation solution: Dilute the antibody with antibody diluent at a dosage of 0.5 μl per 50 μl reaction system, with a final concentration of 0.25 μg / μl. The antibodies used are CD29 (Biolegend, catalog number: 102233); CD68 (Biolegend, catalog number: 137031); CD44 (Biolegend, catalog number: 103045); CD21 / 35 (Biolegend, catalog number: 123427); IgD (Biolegend, catalog number: 405745); CD5 (Biolegend, catalog number: 100637); CD4 (Biolegend, catalog number: 100569); IgM (Biolegend, catalog number: 406535); CD79b (Biolegend, catalog number: 132811); CD169 (Biolegend, catalog number: 142425); CD11c (Biolegend, catalog number: 117355); F4 / 80 (Biolegend, catalog number: 123153); CD27 (Biolegend, catalog number: 124235); CD31 (Biolegend, catalog number: 102437); CD8a (Biolegend, catalog number: 100773).

[0137] III. Experimental procedure: Use the same experimental procedure as in Example 2, where the tissue type is changed to mouse spleen tissue.

[0138] IV. Results and analysis:

[0139] As shown in Figure 5, the expressions of 16 proteins + transcriptomes were successfully detected in mouse spleen tissue.

[0140] Example 4

[0141] I. Experimental tissue: Mouse spleen tissue.

[0142] II. Reagent formulation:

[0143] 1) Blocking solution: In the two groups of experiments, the PolyA concentrations in the blocking solution are 0.5 μM and 10 μM respectively, and the other formulations of the blocking solution are the same as in Table 1 of Example 1

[0144] 2) Antibody diluent: The same as the blocking solution.

[0145] 3) Washing buffer (WB): The same as in Table 2 of Example 1

[0146] 4) Primary antibody incubation solution: Dilute the antibody with antibody diluent at a dosage of 0.5 μl per 50 μl reaction system, with a final concentration of 0.25 μg / μl. The antibodies used are CD4 (Biolegend, catalog number: 100569); CD169 (Biolegend, catalog number: 142425).

[0147] III. Experimental procedures:

[0148] Use the experimental procedures in Example 2, change the tissue type to mouse spleen tissue, and perform sequencing on the proteome alone using a sequencing scheme of 50 bp for the first strand + 15 bp for the second strand.

[0149] IV. Results and analysis:

[0150] As shown in Figure 6, sequencing of the proteome can be achieved when the blocking solution contains 10 μM and 0.5 μM PolyA. In terms of blocking effect, the blocking solution containing 10 μM PolyA is superior to the blocking solution containing 0.5 μM PolyA, showing a certain dose effect.

[0151] Example 5

[0152] I. Experimental tissue: Mouse spleen tissue.

[0153] II. Reagent formulations:

[0154] Use the formulations in the technical solution and divide them into three experimental groups.

[0155] 1) Blocking solution: The same as Table 1 in Example 1

[0156] 2) Antibody diluent: In the first group, the primary antibody incubation solution formulation does not contain protamine DNA and polyA; the second group's primary antibody incubation solution only contains protamine DNA; the third group's primary antibody incubation solution only contains the PolyA group; the fourth group's primary antibody incubation solution contains the protamine DNA and polyA group. The specific component compositions are shown in Table 3.

[0157] Table 3

[0158] 3) Washing buffer (WB): The same as Table 2 in Example 1

[0159] 4) Primary antibody incubation solution: Dilute the antibody with the corresponding antibody diluent in different experimental groups at a dosage of 0.5 μl per 50 μl reaction system, with a final concentration of 0.25 μg / μl. The antibodies used are CD4 (Biolegend, catalog number: 100569); CD45R (Biolegend, catalog number: 103263).

[0160] III. Experimental procedures:

[0161] Using the experimental steps in Example 1 - 9, antibodies were diluted with corresponding antibody diluents in different experimental groups, and the experimental results were judged by the signal - to - noise ratio of immunofluorescence.

[0162] IV. Results and Analysis:

[0163] As shown in Figure 7, the signal - to - noise ratio of the group with 10 μM PolyA added to the antibody diluent was 16.6, and the signal - to - noise ratio of the group with 1 mg / ml salmon sperm was 16.7, which were significantly better than the signal - to - noise ratio of 8.8 in the group without fish sperm DNA and polyA, proving that the addition of fish sperm DNA and polyA can effectively improve the signal - to - noise ratio of detection. In addition, the signal - to - noise ratio of the group with both PolyA and fish sperm DNA added was 37.7, which was higher than the signal - to - noise ratios of the two used alone, proving that the combined use of fish sperm DNA and polyA in the primary antibody diluent can obtain better results.

[0164] Example 6

[0165] I. Experimental tissue: Mouse spleen tissue.

[0166] II. Reagent formula:

[0167] 1) Blocking solution: The same as Table 1 in Example 1

[0168] 2) Antibody diluent: In the first group, there was no fish sperm DNA and polyA in the primary antibody incubation solution formula; in the second group, the primary antibody incubation solution was the same as the blocking solution, containing fish sperm DNA and polyA group. The specific component composition is shown in Table 4.

[0169] Table 4

[0170] 3) Washing buffer (WB): The same as Table 2 in Example 1

[0171] 4) Primary antibody incubation solution: In different experimental groups, antibodies were diluted with corresponding antibody diluents, and the dosage was 0.5 μl / 50 μl reaction system, with a final concentration of 0.25 μg / μl. Among them, the antibodies used were CD4 (Biolegend, catalog number: 100569); CD45R (Biolegend, catalog number: 103263).

[0172] III. Experimental steps:

[0173] Using the experimental steps in Example 1, the signal - to - noise ratio was judged by immunofluorescence and visualization.

[0174] IV. Results and Analysis:

[0175] As shown in Figure 8, when the antibody diluent is the blocking solution, the signal-to-noise ratio (SNR) of IF is 19.2, which is significantly higher than the SNR of 3.7 when the antibody diluent does not contain polyA and salmon sperm DNA. Protein visualization also proves that the effect is better when the antibody diluent is the same as the blocking solution; and it proves that the SNR of protein visualization is very close to the SNR of IF.

[0176] Example VII

[0177] I. Experimental tissue: Mouse spleen tissue.

[0178] II. Reagent formula:

[0179] 1) Blocking solution: The components of the blocking solution are shown in Table 5, and there is no PolyA in the blocking solution.

[0180] Table 5

[0181] 2) Antibody diluent: The same as the blocking solution.

[0182] 3) Washing buffer (WB): The same as Table 2 in Example I

[0183] 4) Primary antibody incubation solution: Dilute the antibody with the antibody diluent, with a dosage of 0.5 μl / 50 μl reaction system and a final concentration of 0.25 μg / μl. Among them, the antibodies used are CD4 (Biolegend, catalog number: 100569); CD45R (Biolegend, catalog number: 103263).

[0184] III. Experimental steps: Use the experimental steps of Example II to sequence the proteome alone.

[0185] IV. Results and analysis: As shown in Figure 9, the absence of PolyA in the blocking solution will lead to experimental failure, and most of the signals are concentrated outside the tissue.

[0186] Example VIII

[0187] I. Experimental tissue: Mouse thymus tissue.

[0188] II. Reagent formula:

[0189] 1) Blocking solution: The same as Table 1 in Example I

[0190] 2) Antibody diluent: The same as the blocking solution

[0191] 3) Washing buffer (WB): The same as Table 2 in Example I

[0192] 4) Primary antibody incubation solution: Dilute the antibody with the corresponding antibody diluent. The dosage is 0.5 μl per 50 μl reaction system, and the final concentration is 0.25 μg / μl. The antibodies used are CD5 (Biolegend, catalog number: 100637); CD8a (Biolegend, catalog number: 100773); CD11c (Biolegend, catalog number: 117355); CD31 (Biolegend, catalog number: 102437).

[0193] III. Experimental procedures:

[0194] For the PFA-fixed group samples, follow the steps described below. For the methanol-fixed group, in steps 1-2, fix with pre-cooled methanol (-20 °C) for 30 min. After the sections are dried, perform the remaining steps according to steps 3-16.

[0195] 1) Take the experimental tissue, prepare frozen tissue sections (10 μm), dry roast for 3 min, and fix in 4% PFA for 10 min.

[0196] 2) Add 400 μl of washing solution WB to a 24-well plate. After the tissue fixation is completed, immediately take out the tissue to prevent it from drying, and immediately add it to the well plate for washing 2 times.

[0197] 3) Add 50 μl of blocking solution to each chip and react at room temperature for 30 min.

[0198] 4) Aspirate the liquid and add 50 μl of primary antibody incubation solution, and react at room temperature for 45 min.

[0199] 5) Aspirate the reaction solution, add 50 μl of washing solution, react for 1 min, repeat this step, and wash a total of 3 times.

[0200] 6) Add secondary antibody incubation solution and react at room temperature for 30 min.

[0201] 7) Aspirate the reaction solution, add 50 μl of washing solution, and react at room temperature for 3 min.

[0202] 8) Aspirate the reaction solution, add 50 μl of washing solution, react for 1 min, repeat this step, wash a total of 3 times, air-dry the chip. After the tissue dries, add 3 μl of glycerol to cover the slide and collect fluorescence images.

[0203] Note: The above steps 1-9 are the experimental procedures for immunofluorescence. The following are the protein capture steps:

[0204] 9) Remove the coverslip, place the chip in 5×SSC solution and react for 5 min to wash away the glycerol.

[0205] 10) Place the chip in a 24-well plate, add 1 ml of 3×SSC, and incubate at 70 °C for 10 min.

[0206] 11) Take out the chip, wash the chip once with 0.1×SSC, add permeabilization reagent, and react at 37°C for 12 min.

[0207] 12) Wash the chip once with 0.1×SSC, add RT mix, and react at 42°C for 2 hours.

[0208] 13) Add 400 μl of tissue removal solution, digest at 55°C for 30 min, remove the tissue removal solution, add 400 μl of digestion solution, and incubate at 55°C for 3 hours.

[0209] 14) Purify cDNA from the digestion solution, amplify the protein library and transcriptome library respectively, prepare DNB, and load it onto the machine for sequencing.

[0210] 15) Among them, for transcriptome sequencing, a sequencing scheme of 50 bp for the first strand + 50 bp for the second strand is adopted, and for proteome sequencing, a sequencing scheme of 50 bp for the first strand + 15 bp for the second strand is adopted.

[0211] 16) Visualize the sequencing results: Data analysis can be carried out through existing bioinformatics analysis processes to obtain specific information and restore the transcriptome and proteome to specific positions on the chip.

[0212] IV. Results and Analysis:

[0213] As shown in Figure 10, it is proved that the process is compatible with methanol fixation and PFA fixation. However, it can also be noted that under the two fixation methods, the detected expression levels of different proteins are different, which may be caused by the fixation differences of different fixation reagents on antigens.

[0214] Example Nine

[0215] I. Experimental Tissue: Mouse liver tissue.

[0216] II. Reagent Formulas:

[0217] 1) Blocking solution: The formula is the same as Table 1 in Example 1, and SSC concentrations of 0.1×, 1×, 3×, 5×, 7×, and 10× are used respectively.

[0218] 2) Antibody dilution solution: The same as the blocking solution.

[0219] 3) Washing buffer (WB): The same as Table 2 in Example 1.

[0220] 4) Primary antibody incubation solution: Dilute the antibody with the corresponding antibody diluent. The dosage is 0.2 μl per 50 μl reaction system, and the final concentration is 0.1 μg / μl. The antibodies used are CD68 (Biolegend, catalog number: 137031), CD4 (Biolegend, catalog number: 100569); CD79b (Biolegend, catalog number: 132811); CD31 (Biolegend, catalog number: 102437); CD11b (Biolegend, catalog number: 101265).

[0221] III. Experimental procedures:

[0222] Refer to Example VIII and operate on the PFA-fixed samples.

[0223] IV. Results and analysis:

[0224] As shown in Figure 11, both too high (10×) or too low (0.1×) ion concentrations in the blocking solution / antibody incubation solution lead to an increase in tissue signal background to a certain extent, such as CD31 or CD11b. However, there is no significant difference in the salt ion concentration of the blocking solution between 1× and 7×, and it has no obvious impact on transcriptome capture.

[0225] Example X

[0226] I. Experimental tissue: Mouse liver tissue.

[0227] II. Reagent formulations:

[0228] 1) Blocking solution: The formulation is the same as Table 1 in Example I, and the pH of the buffer is adjusted to pH = 6, 7, 8, and 9.

[0229] 2) Antibody diluent: The same as the blocking solution.

[0230] 3) Washing buffer (WB): The same as Table 2 in Example I.

[0231] 4) Primary antibody incubation solution: Dilute the antibody with the corresponding antibody diluent. The dosage is 0.2 μl per 50 μl reaction system, and the final concentration is 0.1 μg / μl. The antibodies used are CD68 (Biolegend, catalog number: 137031), CD4 (Biolegend, catalog number: 100569); CD11b (Biolegend, catalog number: 101265), CD11c (Biolegend, catalog number: 117355).

[0232] III. Experimental procedures:

[0233] Refer to Example VIII and operate on the PFA-fixed samples.

[0234] IV. Results and analysis:

[0235] As shown in Figure 12, there is no significant difference in protein detection when the pH value of the blocking solution is between 6 and 9, and there is no obvious impact on transcriptome capture.

[0236] Example XI

[0237] I. Experimental tissue: Mouse liver tissue.

[0238] II. Reagent formula:

[0239] 1) Blocking solution: The formula is the same as Table 1 in Example I.

[0240] 2) Antibody diluent: The same as the blocking solution.

[0241] 3) Washing buffer (WB): The same as Table 2 in Example I.

[0242] 4) Primary antibody incubation solution: Dilute the antibody with the corresponding antibody diluent. The dosage is 0.2 μl / 50 μl reaction system, and the final concentration is 0.1 μg / μl. Among them, the antibodies used are CD68 (Biolegend, catalog number: 137031), CD4 (Biolegend, catalog number: 100569); CD11b (Biolegend, catalog number: 101265), CD11c (Biolegend, catalog number: 117355); CD31 (Biolegend, catalog number: 102437).

[0243] III. Experimental steps:

[0244] Normal process group: Refer to the experimental steps in Example VIII, and operate on the PFA-fixed samples.

[0245] KOH elution group: When reaching step 13 in Example VIII, the tissue digestion solution step was replaced by KOH elution, shortening the process time. The specific operation is as follows:

[0246] 1) After the RT reaction is completed, aspirate and discard the RT reaction solution; add 100 μL of 0.1xSSC solution to the chip for washing;

[0247] 2) Aspirate and discard the 0.1XSSC, add 90 μL of 0.1 M KOH to the chip, ensure that the KOH evenly covers the entire chip, and place it at room temperature for 30 min;

[0248] 3) Aspirate the KOH solution from a corner of the chip, transfer it to a PCR tube, adjust the pH value to 7.4, and purify the protein and transcriptome products respectively for PCR amplification;

[0249] 4) Construct protein libraries and transcriptome libraries respectively, prepare DNB, and perform on-machine sequencing.

[0250] 5) Among them, for transcriptome sequencing, a sequencing scheme of 50 bp for the first strand + 50 bp for the second strand is adopted, and for proteome sequencing, a sequencing scheme of 50 bp for the first strand + 15 bp for the second strand is adopted.

[0251] 6) Visualize the sequencing results: Data analysis can be carried out through existing bioinformatics analysis processes to obtain specific information and restore the transcriptome and proteome to specific positions on the chip.

[0252] IV. Results and analysis:

[0253] As shown in Figure 13, the co-detection of proteins and transcriptome can also be successfully carried out by using the KOH elution method. However, the KOH elution step needs to be further optimized to increase the capture amount of proteins and transcripts.

[0254] Example Twelve

[0255] I. Experimental tissue: Mouse liver tissue.

[0256] II. Reagent formulations:

[0257] 1) Blocking solution: The formulation is the same as that in Table 1 of Example 1, but the concentrations of salmon sperm DNA are 1 mg / ml, 0.5 mg / ml, and 0.1 mg / ml respectively.

[0258] 2) Antibody dilution solution: The same as the blocking solution.

[0259] 3) Washing buffer (WB): The same as that in Table 2 of Example 1.

[0260] 4) Primary antibody incubation solution: Dilute the antibody with the corresponding antibody dilution solution. The dosage is 0.2 μl / 50 μl reaction system, and the final concentration is 0.1 μg / μl. Among them, the antibodies used are CD68 (Biolegend, catalog number: 137031) and CD11b (Biolegend, catalog number: 101265).

[0261] III. Experimental steps:

[0262] For the normal process group, refer to the experimental steps in Example 8 and operate on the PFA-fixed samples.

[0263] IV. Results and analysis:

[0264] As shown in Figure 14, similar visualization effects can be produced when the concentration of salmon sperm DNA is between 0.1 mg / ml and 1 mg / ml.

[0265] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: The multi-omics library construction method and multi-omics detection method in the present application use antibody-conjugated nucleic acids with PolyA and antibody recognition sequence library amplification sequences, and utilize the PolyT structure on the chip to simultaneously capture the PolyA of the antibody-conjugated nucleic acid and the PolyA of the transcriptome, thereby realizing the simultaneous construction of the proteome library and the transcriptome library, and subsequent co-location detection of the proteome and the transcriptome. This method does not require laser photography and only requires simple equipment.

[0266] In addition, when the solution of the present application uses a chip containing PolyT clusters with a 500 nm spacing to capture antibodies containing PolyA and mRNA, by optimizing the blocking solution and antibody hybridization process, the non-specificity of antibody hybridization is reduced, and the capture and library construction of the transcriptome are not affected, thereby realizing co-location detection of the transcriptome and proteome in the whole tissue field of view at the single cell scale, and 30 or more proteins can be detected simultaneously in the best case.

[0267] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. For those skilled in the art, the present invention can have various changes and modifications. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

A method for constructing a multi-omics library, characterized in that, the method comprises: Step S1, placing a sliced sample on a chip with the function of capturing Poly A for protein capture and mRNA capture. Among them, nucleic acid-conjugated antibodies are used for the protein capture. The nucleic acid-conjugated antibodies include antibodies and nucleic acid sequences conjugated to the antibodies. The nucleic acid sequences sequentially include, in the direction away from the antibodies: a PCR linker region, an antibody barcode region, and a Poly A sequence. The chip has Poly T, and the nucleic acid-conjugated antibodies bind to the Poly T on the chip through the Poly A sequence to capture the antigen proteins bound by the antibodies. The mRNA in the sliced sample binds to the Poly T on the chip through Poly A for mRNA capture; Step S2, respectively performing reverse transcription and synthesis of complementary strands on the captured mRNA and the nucleic acid sequences on the nucleic acid-conjugated antibodies bound to the antigens, respectively obtaining cDNA and complementary strands of the nucleic acid sequences on the nucleic acid-conjugated antibodies; Step S3, constructing libraries for the cDNA and the complementary strands of the nucleic acid sequences of the nucleic acid-conjugated antibodies to obtain a transcriptome library and a proteome library of the sliced sample. According to the method described in claim 1, characterized in that, Step S1 includes: Step S11, sealing the chip with a sealing solution to obtain a sealed chip; Step S12, incubating the nucleic acid-conjugated antibodies with the sealed chip to obtain a protein capture chip; Step S13, performing a permeabilization treatment on the protein capture chip to achieve the capture of mRNA and the nucleic acid sequences to obtain a permeabilization-treated chip. According to the method described in claim 2, characterized in that, Step S2 includes: respectively performing reverse transcription and synthesis of complementary strands on the captured mRNA and the nucleic acid sequences on the permeabilization-treated chip to obtain synthesis products, and the synthesis products include cDNA and complementary strands of the nucleic acid sequences; preferably, the temperature of the reverse transcription is 40°C to 44°C, and the time is 1.5 hours to 3 hours. According to the method described in claim 3, characterized in that, Step S3 includes: Step S31, sequentially performing tissue removal and probe digestion on the sliced sample after the reverse transcription and the synthesis of complementary strands on the permeabilization-treated chip to obtain a digestion solution containing the synthesis products; Step S32, separating and purifying the synthesis products from the digestion solution, and respectively amplifying to obtain a proteome library and a transcriptome library. According to the method described in any one of claims 2 to 4, characterized in that, The blocking solution includes: 3×SSC, 5% - 10% (v / v) serum, 0.1% (v / v) Triton-x-100, 0.1 mg / mL - 1 mg / mL salmon sperm DNA, 0.5 μM - 10 μM of 22 bp - 32 bp PolyA, and 5% (v / v) RNase inhibitor; preferably, the concentration of 22 bp - 32 bp PolyA in the blocking solution is 1 μM - 10 μM, more preferably 5 μM - 10 μM, and further preferably 8 μM - 10 μM; preferably, the concentration of salmon sperm DNA in the blocking solution is 0.5 mg / mL - 1 mg / mL. According to the method of claim 5, wherein, in step S12, before incubating the nucleic acid-conjugated antibody with the blocked chip, it further includes a step of diluting the nucleic acid-conjugated antibody with an antibody diluent, wherein the antibody diluent is selected from any one of the following: the blocking solution, a PBS buffer containing PolyA and DNA, or a 2 - 3×SSC buffer containing PolyA and DNA. In the PBS buffer and the SSC buffer, the concentration of PolyA is 0.5 μM - 10 μM, and the concentration of DNA is 0.1 mg / ml - 1 mg / ml. Preferably, the DNA is salmon sperm DNA; preferably, step S12 includes: incubating the nucleic acid-conjugated antibody with the blocked chip so that the nucleic acid-conjugated antibody binds to the antigen to obtain an antibody-antigen complex; placing the antibody-antigen complex in 0.1×SSC buffer at 60°C - 70°C, preferably 65°C - 70°C, and reacting for 5 - 15 min, preferably 10 min, to obtain a protein capture chip. According to the method of claim 2 or 3, wherein, the temperature of the permeabilization treatment is 35°C - 38°C, and the time of the permeabilization treatment is 3 min - 20 min. According to the method of claim 4, wherein, the temperature of tissue removal is 50°C - 60°C, and the time of tissue removal is 20 - 40 min; preferably, the temperature of digestion is 50°C - 60°C, and the time of digestion is 2.5 hours - 4 hours. According to the construction method of claim 1, wherein, The Poly T on the chip is multi-clustered, and the distance between two adjacent clusters is 495 nm to 505 nm; preferably, in the nucleic acid-conjugated antibody, the length of the PCR linker region is 18 bp to 23 bp, the length of the antibody barcode region is 8 bp to 20 bp, and the length of the PolyA is 22 bp to 35 bp; preferably, the antibody part in the nucleic acid-conjugated antibody is selected from any one or more of the following antibodies: CD169 antibody, CD3 antibody, CD8 antibody, CD19 antibody, CD20 antibody, CD21 antibody, CD45R-B220 antibody, CD163 antibody, CD38 antibody, CD11b antibody, CD140a antibody, CD335 antibody, CD371 antibody, CD90.2 antibody, TER-119 antibody, CD274 antibody, CD279 antibody, CD56 antibody, CD14 antibody, CD340 antibody, CD324 antibody, CD38 antibody, CD29 antibody, CD68 antibody, CD44 antibody, CD21 / 35 antibody, IgD antibody, CD5 antibody, CD4 antibody, IgM antibody, CD79b antibody, CD11c antibody, F4 / 80 antibody, CD27 antibody, CD31 antibody and CD8a antibody. A multi-omics detection method, characterized in that, the method includes: constructing a proteome library and a transcriptome library of a sample section to be tested by using the method for constructing a multi-omics library according to any one of claims 1 to 9; sequencing the proteome library and the transcriptome library to obtain the proteome and transcriptome information of the section sample. A multi-omics library construction kit, characterized in that, the kit includes any of the following: Poly A capture chip, nucleic acid-conjugated antibody, blocking solution, tissue permeabilization solution, tissue removal solution and digestion solution; wherein, the blocking solution includes: 3×SSC, 5% to 10% (v / v) serum, 0.1% (v / v) Triton-x-100, 0.1 mg / mL to 1 mg / mL salmon sperm DNA, 0.5 μM to 10 μM of 22 bp to 32 bp PolyA and 5% (v / v) ribonuclease inhibitor. The kit according to claim 11, characterized in that, The concentration of the 22bp - 32bp PolyA in the blocking solution is 1 μM - 10 μM, more preferably 5 μM - 10 μM, and even more preferably 8 μM - 10 μM; preferably, the concentration of the salmon sperm DNA in the blocking solution is 0.5 mg / mL - 1 mg / mL; preferably, the Poly A capture chip refers to a capture chip containing Poly T, and the nucleic acid-conjugated antibody includes an antibody and a nucleic acid sequence conjugated to the antibody. The nucleic acid sequence sequentially includes, in the direction away from the antibody: a PCR adapter region, an antibody barcode region, and a Poly A sequence; preferably, the Poly T on the Poly A capture chip is in multiple clusters, and the distance between adjacent two clusters is 495 nm - 505 nm; preferably, in the nucleic acid-conjugated antibody, the length of the PCR adapter region is 18 - 23 bp, the length of the antibody barcode region is 8 - 20 bp, and the length of the PolyA is 22 bp - 35 bp.

Images