Microarray-based large-area spatial transcriptomic analysis method

Abstract

The present invention relates to a microarray-based large-area spatial transcriptomic analysis method and, specifically, to: a microarray surface treatment method for spatial transcriptomic analysis, the method comprising a step of attaching a poly-T nucleotide sequence by treating the surface of a microarray with a terminal transferase; a microarray for spatial transcriptomic analysis, the microarray being surface-treated by the method; and a sequencing-based large-area spatial transcriptomic analysis method using the surface-treated microarray for spatial transcriptomic analysis.

Description

Microarray-based large-area spatial transcriptome analysis method

[0001] The present invention relates to a microarray-based large-area spatial transcriptome analysis method. Specifically, the present invention relates to a low-cost large-area spatial transcriptome analysis method involving processing a surface probe to enable the use of a microarray for spatial transcription, attaching a tissue section, and performing reverse transcription and sequencing processes.

[0002] Spatial transcriptome technology is a method for analyzing gene expression based on location within a tissue, providing information on cell-to-cell interactions and the influence of the tissue microenvironment on gene expression that was impossible with existing single-cell transcriptome analysis techniques.

[0003] Spatial transcriptome platforms can be divided into FISH-based and NGS sequencing-based methods. FISH-based spatial transcriptome technology is implemented using a targeted capture method, which involves hybridizing gene-binding probes onto tissue sections and then imaging them; while this generally offers high resolution, it has the limitation of restricting the number of genes that can be identified. NGS sequencing-based spatial transcriptome technology involves placing tissue sections onto slides equipped with probes capable of capturing RNA that possess spatial information, and then sequencing the resulting RNA to identify it. Although this method offers relatively lower resolution, it allows for the identification of a large number of genes and the detection of novel RNAs, isoforms, and SNVs.

[0004] The biggest barrier to spatial transcriptomics technology is the problem of high costs. Taking the widely used Visium method as an example, analyzing a single sample with an area of ​​6.5 mm × 6.5 mm costs $2,450, making it difficult to utilize as a primary analytical technique.

[0005] Therefore, there is a need to develop technology that enables efficient spatial transcriptome analysis at a lower cost.

[0006] Accordingly, the inventors of the present invention have completed the present invention by researching the development of a technology capable of enabling low-cost spatial transcriptome analysis, and by utilizing a microarray for RNA capture based on NGS sequencing-based spatial transcriptome technology, thereby developing a method for implementing spatial transcriptome technology capable of large-area observation at low cost.

[0007] Accordingly, the objective of the present invention is to provide a microarray surface treatment method capable of enabling large-area spatial transcriptome analysis at low cost, specifically, to provide a microarray surface treatment method for spatial transcriptome analysis comprising the step of attaching a poly-T base sequence by treating the surface of a microarray with terminal transferase.

[0008] Another objective of the present invention is to provide a microarray for spatial transcriptome analysis surface-treated by the method of the present invention.

[0009] Another objective of the present invention is to provide a sequencing-based large-area spatial transcriptome analysis method characterized by using the surface-treated spatial transcriptome analysis microarray of the present invention.

[0010] Therefore, the present invention provides a microarray surface treatment method for spatial transcriptome analysis, comprising the step of treating the surface of a microarray with terminal transferase to attach a poly-T base sequence.

[0011] In one embodiment of the present invention, a capture area can be created by forming a T-tailing on the surface of the microarray by the terminal transferase treatment.

[0012] In one embodiment of the present invention, the region where the T-tailing is formed can hybridize with a poly-A probe to form a composite.

[0013] In one embodiment of the present invention, the microarray may be an Affymetrix microarray.

[0014] In one embodiment of the present invention, the capture area may capture RNA.

[0015] In addition, the present invention provides a microarray for spatial transcriptome analysis surface-treated by the method of the present invention.

[0016] In addition, the present invention provides a sequencing-based large-area spatial transcriptome analysis method characterized by using the surface-treated spatial transcriptome analysis microarray of the present invention.

[0017] In one embodiment of the present invention, the method comprises: (1) a step of forming a capture area by attaching a poly-T base sequence to the surface of a microarray by treating it with terminal transferase; (2) a step of attaching a tissue to be analyzed for transcriptome analysis to the surface-treated microarray of step (1); (3) a step of releasing mRNA from the tissue by treating the microarray with the attached tissue with collagenase; (4) a step of performing a reverse transcription reaction using reverse transcriptase after capturing the mRNA released in step (3); (5) a step of removing the tissue by treating it with exonuclease I; and (6) a step of synthesizing a secondary strand using a randomomer primer. and (7) the process may be performed by including the step of preparing a library through library PCR and sequencing analysis using the secondary strand synthesized in step (6).

[0018] In this invention, a method for treating the surface of a microarray to enable its use in spatial transcriptome analysis has been developed by modifying the conventional use of the microarray. When spatial transcriptome analysis is performed using the surface-treated microarray of this invention, it is possible to reduce costs compared to existing technologies and to perform large-area spatial transcriptome analysis with high resolution.

[0019] Figure 1 shows a schematic diagram of the pretreatment for microarray surface treatment and capture region generation according to the present invention, and mRNA capture and secondary strand synthesis using the pretreated microarray.

[0020] Figure 2 shows the results of confirming that mRNA transcripts with an A tail can be detected using a pre-processed microarray according to the present invention.

[0021] Figure 3 shows the results confirming that T-tailing proceeded on the ssDNA substrate by terminal transferase (TdT enzyme) (left) and the results confirming that a probe complex was formed by hybridizing the poly-A probe to the T-tail generated by the terminal transferase when reacted with a fluorescently labeled poly-A probe.

[0022] The present invention is characterized by the development of a method that enables large-area spatial transcriptome analysis at a low cost compared to conventionally developed spatial transcriptome platforms.

[0023] Specifically, the inventors have devised a surface treatment method for a microarray for large-area spatial transcriptome analysis. The surface treatment method for a microarray for spatial transcriptome analysis according to the present invention includes the step of attaching a poly-T base sequence to the surface of the microarray by treating it with a terminal transferase.

[0024] The above-mentioned terminal transferase is also known as terminal deoxynucleotide transferase (TdT) and catalyzes the addition of nucleotides to the 3' end of a DNA molecule. Unlike most DNA polymerases, nucleotide addition is possible without the aid of a template strand. The preferred substrate is the 3'-protrusion, but nucleotides can also be added to the blunt or indented 3' end.

[0025] In the present invention, T-tailing is formed on the surface of the microarray by the terminal transferase treatment, thereby creating a capture area. The capture area corresponds to a region capable of capturing RNA expressed in the sample (cell or tissue) to be analyzed.

[0026] The region where the above T-tailing is formed can form a complex through hybridization of complementary base sequences when a poly-A probe is added.

[0027] In the present invention, a microarray surface-treated to form a T-tailing has a poly-A-tail at the 3' end of RNA, which is a transcript product, so that RNA can be captured.

[0028] The microarray that can be used in the present invention is an Affymetrix microarray, which is a photolithography-based DNA microarray. Oligonucleotide probes capable of hybridizing with target nucleic acids are attached to the surface according to location and type. After placing a sample in the microarray cartridge and hybridizing it, the signal according to the location can be checked through a scanner to confirm gene or miRNA expression, etc. Commercially available Affymetrix microarrays have oligonucleotide probes of known sequences attached to designated locations, the feature size of the probe is 11 μm, and the size of the glass to which the probe is attached is approximately 10 mm × 10 mm.

[0029] In the present invention, a microarray surface treatment method was devised as described above to enable the use of Affymetrix microarrays for RNA capture based on Next Generation Sequencing (NGS) sequencing-based spatial transcriptome technology.

[0030] The Affymetrix microarray used in this invention has an area of ​​approximately 10 mm × 10 mm; the cost per unit is $200, the reagent price is $100, and the sequencing cost is around $500. That is, 1 mm 2 It is possible to implement spatial transference technology with a resolution of 11 µm for about $8.

[0031] Meanwhile, the cost of Visium technology, a conventional spatial transcriptome analysis technique, is 1 mm 2 The cost is approximately $58, and the resolution is about 100 μm. Therefore, the method provided by the present invention exhibits superior resolution compared to existing methods and has the excellent effect of reducing costs to about 1 / 7.

[0032] In addition, the present invention can provide a microarray for spatial transcriptome analysis surface-treated by the method of the present invention.

[0033] As previously described, the above surface treatment refers to the formation of T-tailing on the surface of a microarray by terminal transferase treatment.

[0034] Furthermore, the present invention can provide a sequencing-based large-area spatial transcriptome analysis method characterized by using the surface-treated spatial transcriptome analysis microarray of the present invention.

[0035] Preferably, the method comprises: (1) a step of forming a capture area by attaching a poly-T base sequence to the surface of a microarray by treating it with terminal transferase; (2) a step of attaching a tissue to be analyzed for transcriptome analysis to the surface-treated microarray of step (1); (3) a step of releasing mRNA from the tissue by treating the microarray with the attached tissue with collagenase; (4) a step of performing a reverse transcription reaction using reverse transcriptase after capturing the mRNA released in step (3); (5) a step of removing the tissue by treating it with exonuclease I; and (6) a step of synthesizing a secondary strand using a randomomer primer. and (7) the process may be performed by including the step of preparing a library through library PCR and sequencing analysis using the secondary strand synthesized in step (6).

[0036] A specific method for sequencing-based large-area spatial transcriptome analysis using a surface-treated microarray of the present invention is described in the following examples.

[0037] The sequencing-based large-area spatial transcriptome analysis method using the surface-treated microarray provided in this invention enables the analysis of tissue sections of sizes that are impossible to image on currently commercialized spatial transcriptome platforms, thereby allowing for spatial transcriptome analysis of large-area samples.

[0038] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited to these examples.

[0039]

[0040] <Example 1>

[0041] Oligomer Design

[0042] <1-1> Primer Design for Secondary Strand Synthesis

[0043] After tissue section attachment and reverse transcription, primers for secondary strand synthesis were designed using the sequences in Table 1 below. Randomers possess 9 N sequences, which bind complementarily to the reversed strand and are used as primers for secondary strand synthesis. The 9 N sequences function as Unique Molecular Identifiers (UMIs) during the subsequent sequencing process. The sequences on the 5' side function as read primers during library construction.

[0044] Name5'-Sequence-3'ModificationSequence NumberRandomerTCAGACGTGTGCTCTTCCGATCTNNNNNNNNN-1

[0045]

[0046] <1-2> Primer Design for Sequencing Library Construction

[0047] After the second strand synthesis, primers for constructing a library using the obtained reaction product were designed as shown in Table 2 below. A first-round library PCR was performed using RPEPCR*F and RPEPCR*R primers, and a second-round library PCR was performed using P5 Fwd and P7 Rev indexing primers to construct a sequencing library.

[0048] Name5'-Sequence-3'SEQ ID NO:ModificationRPEPCR*FTCT TTC CCT ACA CGA CGC*T*C2Internal phosphorothioate (*marked site)RPEPCR*RTCA GAC GTG TGC TCT TCC*G*A3P5 FwdAATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCT*T*C4P7 Rev indexingCAAGCAGAAGACGGCATACGAGAT [8-mer index sequence] GTGACTGGA GTTCAGACG TGTGCTCTTCC*G*A5

[0049]

[0050] <Example 2>

[0051] Microarray surface treatment for spatial transcriptome implementation

[0052] A capture area was formed by attaching a poly-T sequence to the surface of the Affymetrix microarray using terminal transferase, and the specific process is as follows.

[0053] First, a T-tailing mixture containing the ingredients of Table 3 below was prepared and transferred to a new PCR tube.

[0054] T-tailing mixed solutionComponentsVolume10

[0055] After thoroughly mixing the reactants, they were loaded into an Affymetrix microarray cartridge, and the End repair and A-tailing reactions were carried out under the conditions of Table 4 below.

[0056] End repair and A-tailing reaction conditions Temperature Time 37°C Overnight

[0057] Then, the microarray cartridge was washed three times with 0.1M Tris buffer (pH 7.5), washed three times with ultrapure purified water, air-dried, and stored at 4°C.

[0058] Through this process, a poly-T sequence was attached to the surface of the microarray, and the results were confirmed by hybridizing with a fluorescently labeled poly-A probe (Atto 565-AAAAAAAAAAAAAAAAAAAAAAAAAAA) and imaging with an EVOS 7000 instrument.

[0059] As a result of the analysis, as shown in Figure 2, T-tailing occurred on the P side of the microarray treated with terminal transferase, and a fluorescent signal was observed due to hybridization of the fluorescently labeled poly-A probe, while some non-specific weak fluorescent signals were observed on the N side that was not treated with the terminal transferase effect.

[0060] In addition, the inventors analyzed whether T-tailing occurs on the ssDNA substrate by terminal transferase (TdT enzyme), and as shown in Figure 3, confirmed that when reacted with a fluorescently labeled poly-A probe, the poly-A probe hybridizes to the T-tail generated by the terminal transferase to form a probe complex.

[0061]

[0062] Through the experiment of Example 2 above, a microarray usable for spatial transcription was prepared by treating the surface of the microarray with terminal transferase to form a T tail. Next, the inventors performed the experiments of the following examples to confirm whether the microarray prepared in Example 2 above could detect mRNA transcripts having an A tail.

[0063]

[0064] <Example 3>

[0065] Tissue section attachment to microarray and imaging analysis

[0066] After attaching tissue sections to the microarray prepared in Example 2 above, the tissue was stained and imaging analysis was performed.

[0067]

[0068] <3-1> Tissue Cutting and Attachment

[0069] A metal beaker was filled halfway with isopentane and placed inside a liquid nitrogen tank. The tissue sample was wrapped in OCT compound and transferred to a cryomold. The cryomold containing the tissue sample was transferred to isopentane and left until the OCT compound hardened and turned opaque white. Once the tissue was frozen, it was removed and placed on dry ice for at least 30 minutes, then transferred to a cryostat at -20°C and left for at least 20 minutes. Subsequently, the tissue was sectioned to a thickness of 10 μm in the cryostat and attached to a microarray. Then, 1 mL of 4% paraformaldehyde was loaded and left for 10 minutes, after which it was washed three times with 1 mL of PBS.

[0070]

[0071] <3-2> Tissue H&E Staining and Imaging Analysis

[0072] 50 μL of isopropanol was loaded onto the microarray with attached tissue sections, left for 1 minute, and then wiped off. 50 μL of hematoxylin was evenly loaded and left for 7 minutes. Subsequently, the hematoxylin was removed by washing with 1 mL of ultrapure water. Next, 50 μL of Bluing Buffer was loaded and left for 2 minutes. Afterward, the hematoxylin was removed by washing with 1 mL of ultrapure water. 50 μL of eosin staining solution was loaded and aspirated with a pipette. Then, the process was repeated three times by washing with 1 mL of ultrapure water. Afterward, the microarray was dried, 20 μL of 85% glycerol was loaded, and the chip was transferred to a slide glass. The tissue was imaged and analyzed using an inverted optical microscope, the glycerol was removed with 80% ethanol, and the sample was dried.

[0073]

[0074] <Example 4>

[0075] mRNA capture and reverse transcription

[0076] The following mRNA capture and reverse transcription reactions were performed using a microarray with tissue sections attached in <Example 3> above.

[0077]

[0078] <4-1> mRNA release

[0079] 50 μL of collagenase working solution was loaded onto the microarray to which the tissue section prepared in <Example 3> was attached, and the mixture was left at 37 °C for 20 minutes. Then, 50 μL of pepsin working solution was loaded onto the microarray and left at 37 °C for 10 minutes.

[0080] The collagenase working solution is a solution composed of 1 μl of Collagenase I stock solution (50 units / μl) and 249 μl of HBSS solution, and the pepsin working solution is a solution composed of 1 μl of Pepsin stock solution (0.1 g / mL) and 198 μl of 0.1 M HCl.

[0081]

[0082] <4-2> mRNA Capture and Reverse Transcription

[0083] 50 μL of RT Equilibration Solution was loaded onto the microarray after the process of <4-1> was completed and left at room temperature for 10 minutes. The microarray was loaded with 50 μL of RT solution and left overnight at a temperature of 42°C in a humidity-maintained reactor. Afterward, the RT solution was removed.

[0084] The RT equilibrium solution is a solution composed of 155 μL of Ultrapure Water, 40 μL of Maxima RT buffer (5X), and 5 μL of RNase Inhibitor (40 units / μl), and the RT solution is a solution composed of 85 μL of Ultrapure Water, 40 μL of Maxima RT buffer (5X), 40 μL of 20% Ficoll PM 400, 20 μL of 10 mM dNTP Mix, 5 μL of RNase Inhibitor (40 units / μl), and 10 μL of Maxima H Minus Reverse Transcriptase.

[0085]

[0086] <4-3> Tissue Removal

[0087] 50 μL of Exonuclease I solution was loaded onto the microarray and incubated at 37 °C for 45 minutes to remove all unreacted capture probes. Subsequently, 50 μL of Tissue Digestion Buffer was loaded and incubated at 37 °C for 45 minutes to remove tissue regions excluding mRNA. Then, the process of loading and removing 200 μL of ultrapure purified water for 1 minute was repeated three times, the process of loading and removing 200 μL of 0.1N NaOH for 5 minutes was repeated three times, the process of loading and removing 200 μL of 0.1M Tris buffer (pH 7.5) for 1 minute was repeated three times, and the process of loading and removing 200 μL of ultrapure purified water for 1 minute was repeated three times.

[0088] The above Exonuclease I solution is a solution composed of 170 μL of Ultrapure Water, 10 μL of Exonuclease I (20 units / μl) and 20 μL of Exonuclease I buffer (10X), and the above Tissue Digestion Buffer is a solution composed of 126 μL of Ultrapure Water, 20 μL of 1 M Tris-HCl pH 8.0, 20 μL of 2 M NaCl, 20 μL of 20% SDS, 10 μL of 0.1 M EDTA and 4 μL of Proteinase K (0.8 units / μl).

[0089]

[0090] <4-4> Synthesis and elution of the second strand

[0091] Next, 50 μL of Random Priming and Extension mix was loaded and reacted at 37 °C for 2 hours to synthesize the secondary strand. Subsequently, the sample was washed three times with 200 μL of ultrapure purified water, loaded with 50 μL of 0.1N NaOH, and left at room temperature for 5 minutes. The loaded NaOH solution was recovered and transferred to a 1.5 mL LoBind tube. The process of loading 50 μL of 0.1N NaOH, leaving at room temperature for 5 minutes, and then recovering the solution was repeated one more time. The synthesized secondary strand was eluted through this process. Then, 50 μL of Zymogen neutralization buffer was added to 100 μL of the NaOH solution and mixed by pipetting. The obtained secondary strand was purified using an AMPure XP magnetic bead.

[0092] The above random priming and extension mix is ​​a solution composed of 120 μL of Ultrapure Water, 20 μL of NEBuffer 2 (10X), 20 μL of 100 μM Randomer, 20 μL of 10 mM dNTP Mix, and 20 μL of Klenow fragment (3'→5' exo-) (5 units / μl).

[0093]

[0094] <Example 5>

[0095] Sequencing library construction and sequencing

[0096] <5-1> 1st-Round Library PCR

[0097] After preparing the library amplification PCR mix, 50 μL was added to each PCR tube, and the reaction was performed under the following conditions. The library amplification PCR mix consisted of 16 μL of Ultrapure Water, 80 μL of Purified second strand solution, 100 μL of KAPA HiFi HotStart ReadyMix (2X), 2 μL of RPEPCR*F primer (100 μM), and 2 μL of RPEPCR*R primer (100 μM). Subsequently, the PCR product obtained was purified using AMPure XP magnetic beads.

[0098] 1st-Round Library PCR Conditions Step (15 cycles) Temperature (°C) Time Denaturation 953 min Annealing 601 min Extension 721 min Final extension 722 min Hold 4 Hold

[0099]

[0100] <5-2> 2nd-Round Library PCR

[0101] 40 μL of the Indexing PCR reaction solution was added to each PCR tube, and the reaction was carried out as follows. The Indexing PCR reaction solution is a solution composed of 8 μL of Ultrapure Water, 100 μL of KAPA HiFi HotStart ReadyMix (2X), 40 μL of 2 nM purified PCR product, 16 μL of P5 Fwd primer (10 μM), and 16 μL of P7 Rev indexing primer (10 μM).

[0102] 2nd Round Library PCR Conditions Step (9 cycles) Temperature (°C) Time Denaturation 95 30 s Annealing 60 30 s Extension 72 30 s Final extension 72 2 min Hold 4 Hold

[0103] Once the PCR reaction was complete, the PCR product obtained was purified using Zymogen DNA Clean and the Concentration-25 kit. The indexing PCR product was subjected to electrophoresis using a 2% agarose gel, and bands ranging in size from 400 to 800 bp were separated. The PCR product within the gel was recovered using the Zymogen Gel DNA Recovery kit, and the PCR product obtained was purified using AMPure XP magnetic beads.

[0104]

[0105] <5-3> Library Sequencing

[0106] Next, sequencing was performed according to the experimental method of the Illumina PE150 manufacturer to obtain gene expression information including spatial information.

[0107]

[0108] Through the method described above, the inventors developed a method for implementing sequencing-based spatial transcriptome technology using Affymetrix microarrays, and confirmed that when the method developed in the present invention is applied, analysis is possible at a much lower cost compared to existing spatial transcriptome analysis methods, and it was found that when the method of the present invention is applied, the observable area is on the wider side compared to existing spatial transcription platform technology.

[0109]

[0110] The present invention has been described above with reference to its preferred embodiments. Those skilled in the art will understand that the present invention may be embodied in modified forms without departing from the essential characteristics of the invention. Therefore, the disclosed embodiments should be considered in an illustrative rather than a restrictive sense. The scope of the invention is defined by the claims, not by the foregoing description, and all variations within the scope of the claims should be interpreted as being included in the invention.

Claims

1. A step comprising attaching a poly-T base sequence to the surface of a microarray by treating it with terminal transferase, Microarray surface treatment method for spatial transcriptome analysis.

2. In Paragraph 1, A microarray surface treatment method for spatial transcriptome analysis, characterized by forming T-tailing on the surface of the microarray by the above-mentioned terminal transferase treatment to create a capture area.

3. In Paragraph 2, A microarray surface treatment method for spatial transcriptome analysis, characterized in that the region where T-tailing is formed hybridizes with a poly-A probe to form a complex.

4. In Paragraph 1, A microarray surface treatment method for spatial transcriptome analysis, characterized in that the microarray is an Affymetrix microarray.

5. In Paragraph 2, A microarray surface treatment method for spatial transcriptome analysis, wherein the above-mentioned capture area is characterized by capturing RNA.

6. A microarray for spatial transcriptome analysis surface-treated by the method of any one of paragraphs 1 to 5.

7. A sequencing-based large-area spatial transcriptome analysis method characterized by using the surface-treated spatial transcriptome analysis microarray of claim 6.

8. In Paragraph 7, The above method is, (1) A step of forming a capture area by attaching a poly-T base sequence to the surface of a microarray by treating it with terminal transferase, (2) A step of attaching a tissue to be analyzed in a transcriptome to the surface-treated microarray of step (1) above; (3) A step of releasing mRNA from the tissue by treating the microarray to which the tissue is attached with collagenase; (4) A step of capturing the mRNA released in step (3) above, and then performing a reverse transcription reaction using reverse transcriptase; (5) A step of removing tissue by treating with exonuclease I; (6) A step of synthesizing a secondary strand using a randomer primer; and (7) A step comprising preparing a library by performing library PCR and sequencing analysis using the secondary strand synthesized in step (6) above, Sequencing-based large-area spatial transcriptome analysis method.

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