Improved nucleic acid capture method

Abstract

The improved nucleic acid capturing method comprises the following steps: hybridizing a random probe and an oligonucleotide chain fixed on a capturing chip through a splint oligonucleotide, reversely transcribing RNA (Ribonucleic Acid) in a sample captured by the random probe into cDNA (Complementary Deoxyribonucleic Acid), connecting the cDNA to the oligonucleotide chain, and carrying out reverse transcription and connection in the same reaction system. In the process, RNA is subjected to reverse transcription to form cDNA, and meanwhile, the cDNA is connected to an oligonucleotide chain fixed on a chip by virtue of fixed oligonucleotide sequences at two ends hybridized by splint. Through the method, the number of captured genes can be increased, the loss of cDNA (complementary deoxyribonucleic acid) is reduced, the original three steps are combined into one step, and the duration of the process can be further shortened.

Description

An improved nucleic acid capture method Technical Field

[0001] The present invention belongs to the field of genes, and in particular relates to an improved nucleic acid capture method. Background Art

[0002] Cells within an organism perform specific functions within their spatial locations, and the gene expression of each cell is closely related to the physiological or pathological processes it controls. Spatiotemporal transcriptomics technology can analyze gene expression in situ within tissues, enabling the study of developmental and disease processes.

[0003] The currently used spatiotemporal transcription technology is to reverse transcribe the RNA captured in the tissue to obtain stable cDNA, and then connect the cDNA in situ to the capture chip, so as to fix the spatial position of the cDNA, and then obtain the expression of the gene at a certain spatial position through sequencing.

[0004] However, this method captures far fewer genes than expected, resulting in many genes being missed, which in turn results in spatiotemporal transcriptome analysis that cannot accurately reflect the true gene expression profile of the tissue. Therefore, a method that can capture more genes is urgently needed to more accurately reflect the true gene expression profile of the tissue.

[0005] Summary of the Invention

[0006] The present invention mainly addresses the problem of cDNA loss in the prior art and provides an improved nucleic acid capture method. Specifically, the present invention combines RT and T4 ligase into the same step. In this step, RT enzyme and T4 ligase are added at the same time, and the reaction is carried out at 37°C for 3-5 hours. In this process, while RNA is reverse transcribed into cDNA, the cDNA is connected to the oligonucleotide chain fixed on the chip through splint hybridization of the fixed oligonucleotide sequences at both ends. Through this method, the number of captured genes can be greatly increased, the loss of cDNA can be reduced, and the duration of the process can be greatly shortened.

[0007] First, place tissue sections (frozen or paraffin samples) on the capture chip surface, and then fix and permeabilize the sections (paraffin samples must first be dewaxed and decrosslinked).

[0008] While the tissue is being fixed, random probes such as 6N and splint are hybridized in a 5×SSC solution at 55° C. for 10 minutes ( ① in FIG. 2 ).

[0009] After tissue permeabilization is completed, the hybridized primer hybridization mixture is added to the chip surface and hybridized at room temperature in a 5×SSC environment for 15-30 minutes. During this process, the random probes in the primer hybridization mixture, such as the 6N part, will capture the RNA in the tissue (② in Figure 2).

[0010] Then, the RT and T4 ligation reaction solution is added to the chip surface and reacted at 37°C for 3-5 hours, so that the RT and T4 ligation reactions proceed simultaneously, that is, the cDNA is fixed on the capture chip through T4 ligation at the same time as RT (③ in Figure 2).

[0011] The operational flow chart of the method of the present application (RT+T4 connection performed simultaneously) and the method of comparative example 1 (RT first, then T4 connection) is shown in Figure 3. The operational flow details of the method of Example 1 are as follows (combined with Figures 2 and 3): slice the fresh tissue embedding block, attach the slices to the capture chip, bake the slices at 37°C for 3 minutes (dewax and decrosslink the paraffin tissue slices), and then fix the chip with the tissue in -20°C methanol for 30 minutes. The subsequent processes of the two tissue slices are the same: while the tissue is fixed with methanol, a random probe (containing 6N) with a fixed oligonucleotide sequence at the 5' end and splint are mixed in a molar ratio of 1:1 with 5×SSC containing RNase inhibitor (RI) to a final concentration of 0.1 μM, and placed in 55°C for hybridization for 10 minutes to hybridize the splint and the fixed oligonucleotide sequence at the 5' end of the random probe to form a hybridization probe. The chip with the tissue section attached is fixed in methanol, removed, and permeabilized at 37°C for 20 minutes. The chip is then removed and washed once with 5× SSC supplemented with RI. A mixture of pre-hybridized random probes and splint is added to allow hybridization between the random probes, such as 6N, and the RNA within the tissue. Hybridization is carried out at room temperature for 15 minutes. The chip is then washed once with 5× SSC supplemented with RI to remove any random probes that have not hybridized to the RNA. The liquid from the chip surface is aspirated, and a premix containing reverse transcriptase (RT) and T4 ligase is added. The chip is incubated at 37°C for at least 3 hours, or up to overnight. After the reaction, the chip surface is rinsed with nuclease-free purified water. Tissue removal solution is then added, and tissue removal is carried out at 55°C for 10 minutes (20 minutes for paraffin tissue). After tissue removal, a cDNA release mix is ​​added and incubated at 55°C for 3 hours to release the cDNA into solution. The resulting cDNA is then purified using magnetic beads and amplified by PCR to generate a cDNA library. 20 ng of cDNA library was taken and sheared with Tn5 shear enzyme to obtain a sequencing library for second-generation high-throughput sequencing.

[0012] The method provided by this invention combines reverse transcriptase and T4 ligase in a single system. During this process, RNA is reverse transcribed into cDNA while the cDNA is ligated to oligonucleotide chains immobilized on the chip through splint hybridization between random probes and oligonucleotide chains on the chip. This method significantly increases the number of genes captured, reduces cDNA loss, and further significantly shortens the process time.

[0013] In order to solve the defects of the prior art, the first aspect of the present invention provides a method for capturing nucleic acid, which comprises the following steps:

[0014] The random probe and the oligonucleotide chain fixed on the capture chip are hybridized by splint oligonucleotides, the RNA in the sample captured by the random probe is reverse transcribed into cDNA, and the cDNA is connected to the oligonucleotide chain, and the reverse transcription and the connection are carried out in the same reaction system.

[0015] In certain embodiments, it includes the following steps:

[0016] (1) hybridizing the random probe and the splint sequence; wherein the 5' end of the random probe is partially complementary to the 5' end of the splint sequence;

[0017] (2) the random probe hybridizes with RNA in the sample; wherein the 3' end of the random probe is partially complementary to the 3' end of the RNA;

[0018] (3) The splint sequence hybridizes to the fixed oligonucleotide sequence.

[0019] Among them, (1), (2) and (3) have no order and can occur simultaneously in the same reaction system.

[0020] Preferably, the oligonucleotide sequence is immobilized on a capture chip;

[0021] (4) In the same reaction system, cDNA is synthesized using the RNA in the complex formed by the simultaneous hybridization in (1), (2) and (3) as a template, and the fixed oligonucleotide sequence and the random probe on the complex are connected, so that the fixed oligonucleotide sequence, the random probe and the cDNA form a long single-stranded nucleotide containing cDNA in the 5' to 3' direction.

[0022] There is no temporal order for the above steps (3) and (4), and they can be performed simultaneously.

[0023] In certain embodiments, in step (1), the random probe comprises 6-20N;

[0024] In step (3), the capture chip is placed at 30-45° C., for example, 37° C., for reaction for 3-24 hours, for example, 3-5 hours;

[0025] In the reaction system of step (4), reverse transcriptase and DNA ligase are present simultaneously.

[0026] Preferably, the sample comprises a tissue sample.

[0027] In some embodiments, the steps further include:

[0028] (5) Absorbing the liquid on the surface of the capture chip, adding a mixture 1 of reverse transcriptase and T4 ligase, placing the capture chip in an incubator, and reacting for 3-24 hours; the T4 ligase is, for example, a high-temperature resistant T4 ligase or a high-salt resistant T4 ligase.

[0029] In certain embodiments, the mixture 1 comprises reverse transcription reagents, 3-25 mM MgCl2, 1-50 mM DTT, and ≥1 mM ATP;

[0030] The volume ratio of the reverse transcriptase to the T4 ligase is 2:1 or 1:1; and / or,

[0031] The temperature of the incubator is 35-42°C. The reaction time can be prolonged appropriately at a low temperature, for example, the reaction can be carried out at 35°C for 24 hours.

[0032] In certain embodiments, the pH of the mixed solution is 8.3.

[0033] The optimal reaction buffer for reverse transcriptase consists of 3mM MgCl2, 10mM DTT, pH 8.3 at 25°C, and an optimal reaction temperature of 42°C. The optimal reaction buffer for T4 ligase consists of 10mM MgCl2, 1mM ATP, 10mM DTT, pH 7.5 at 25°C, and an optimal reaction temperature of 16°C. During the experimental design process, considering the differences in the optimal reaction buffers and reaction temperatures for the two enzymes, we tested capture performance under different buffer compositions and reaction temperatures. We found that using the reverse transcriptase reaction buffer, adding 1mM ATP, and using a reaction temperature of 37°C resulted in better capture results.

[0034] In some embodiments, before step (5), the method further includes:

[0035] (i) While the tissue section is being fixed to the capture chip, a probe hybridization solution is prepared, mixed with 5×SSC, and hybridized in an incubator at 50-60° C., for example, 55° C., for 5-30 minutes, for example, 10 minutes, to form a primer hybridization mixture. The mixture is then removed from the incubator, cooled, and 2-15% by volume, for example, 5% by volume, of an RNase inhibitor is added;

[0036] (ii) removing the capture chip, removing the surface liquid, washing the capture chip with mixed solution 2, adding the primer hybridization mixture, hybridizing at room temperature for 15 to 60 minutes, and then washing again with mixed solution 2;

[0037] The mixed solution 2 is 5×SSC and RNase inhibitor;

[0038] The probe hybridization solution contains random probes and splint.

[0039] The above steps (i) and (ii) are performed before step (5) and after step (4).

[0040] In certain embodiments, the tissue section is a section of frozen OCT tissue;

[0041] The probe hybridization solution is a random probe and splint with a molar ratio of 1:1;

[0042] The cooling method is to place the mixture on ice and / or place the mixture at room temperature until the mixture cools to room temperature, and / or,

[0043] The volume ratio of 5×SSC to RNase inhibitor in the mixed solution 2 is (5-30):1, for example, 19:1.

[0044] In certain embodiments, the sequence of the random probe is shown as SEQ ID NO: 2; the sequence of the splint is shown as SEQ ID NO: 3.

[0045] In some embodiments, the method further comprises the following steps:

[0046] (a) after completing step (ii), removing the sample tissue on the capture chip;

[0047] (b) recovering cDNA from the capture chip; optionally, further comprising steps of cDNA amplification and purification.

[0048] In certain embodiments, the capture chip includes a capture probe, wherein the capture probe includes spatial barcode position information and a fixed oligonucleotide sequence; the fixed oligonucleotide sequence hybridizes with the 3' end of the splint. Furthermore, the fixed oligonucleotide sequence can be connected to the 5' end of the random probe.

[0049] In certain embodiments, the spatial barcode position information is Nm, the fixed oligonucleotide sequence is SEQ ID NO: 1, and the capture probe is 5'Nm-SEQ ID NO: 1-3', wherein m is 8-30.

[0050] In certain embodiments, said m≥25.

[0051] The capture chip also includes an immobilized oligonucleotide sequence', which is used for PCR amplification of the cDNA library and sequencing of one strand of the library.

[0052] A second aspect of the present invention provides a high-throughput sequencing method, comprising the following steps:

[0053] (I) obtaining the long single-stranded nucleotide sequence containing cDNA to be sequenced according to the method of the first aspect of the present invention;

[0054] (II) Construction of cDNA library;

[0055] (III) Perform sequencing and analyze the obtained data.

[0056] In certain embodiments, (I) further includes the steps of cDNA shearing, amplification, and purification; and / or, in (II), the cDNA library is circularized to obtain a circularized DNB library.

[0057] On the basis of conforming to the common sense in this field, the above-mentioned preferred conditions can be arbitrarily combined to obtain the preferred embodiments of the present invention.

[0058] The reagents and raw materials used in the present invention are commercially available.

[0059] The positive progress effect of the present invention is:

[0060] The method of the present invention combines reverse transcriptase and T4 ligase in the same system. During this process, RNA is reverse transcribed into cDNA, and the cDNA is simultaneously ligated to oligonucleotide chains immobilized on the chip through splint hybridization between random probes and oligonucleotide chains on the chip. This method combines the original three steps into one, increasing the amount of gene capture, reducing cDNA loss, and significantly shortening the process duration. BRIEF DESCRIPTION OF THE DRAWINGS

[0061] FIG1 is a schematic diagram of the spatiotemporal transcriptome technology based on random probe capture of RNA in Comparative Example 1.

[0062] FIG2 is a schematic diagram showing the principle of the improved RT and T4 ligation method of the present application (the arrow direction is the 3′ end of the oligonucleotide chain).

[0063] FIG3 is a comparison diagram of the operation flow charts of Comparative Example 1 (RT followed by T4 connection) and Example 1 (RT+T4 connection).

[0064] FIG4 shows the principle of the method of connecting T4 first and then RT.

[0065] FIG5 is a schematic diagram showing the hybridization of a single splint with an oligonucleotide chain on a chip.

[0066] FIG6 is a comparison diagram of the gene capture effects of the RT+T4 ligation method of Example 1 and the RT-first-then-T4 ligation method of Comparative Example 1.

[0067] FIG7 shows the capture effects of the T4 ligation followed by RT method in Comparative Example 2 and the variable temperature hybridization method in Example 4. DETAILED DESCRIPTION

[0068] Example 1 RT+T4 connection method

[0069] 1.1 Frozen sample sectioning

[0070] Precool the cryostat cabinet (-20°C) and the specimen head (-10°C to -15°C). Place brushes, blades, and other laboratory tools in the -20°C cabinet. Remove the frozen OCT (Optimal Cutting Temperature Compound)-embedded tissue block from the -80°C freezer and equilibrate it in the cryostat. Use room-temperature liquid OCT to secure the tissue block to the specimen holder and freeze until the OCT solidifies. As needed, trim the tissue block with a precooled blade before sectioning.

[0071] 1.2 RNA quality control

[0072] Ten to twenty 10-μm-thick tissue sections were cut and placed in 1.5-ml EP tubes precooled at −20°C. Total RNA was extracted using the RNeasy Mini Kit (QIAGEN) and RNA quality was tested using the Agilent RNA 6000 Pico Kit (Agilent). Samples with a RIN ≥ 7 could be used in subsequent experiments.

[0073] 1.3 Chip processing and tissue patching

[0074] Carefully pick up the capture chip with tweezers and place it in a new 24-well plate. The capture chip contains a capture probe, which includes spatial barcode position information and a fixed oligonucleotide sequence: 5'-N 25 -TTGTCTTCCTAAGACCGCTTGG-3' (the underlined part is SEQ ID NO: 1, N 25(N represents 25 consecutive Ns). Wash the chip twice with 400 μl of 0.1× SSC, rinse thoroughly with nuclease-free water (NF-H2O), and bake the chip at 37°C to dry. Cut a 10 μm thick slice of frozen OCT tissue, flatten it, and grasp the capture chip with forceps. Quickly approach the frozen slice to adhere the tissue slice to the chip. Then bake the chip with the tissue slice at 37°C for 3–5 minutes.

[0075] 1.4 Tissue section fixation and permeabilization

[0076] The baked chip was fixed in a -20°C pre-cooled methanol solution for 30 minutes. After removal, the methanol was dried and permeabilization reagent (permeabilization enzyme (Stereo-seq Transcriptome Kit T, Catalog No.: 101KT114; PR Enzyme 1000028500, PR Rinse Buffer 1000033684) mixed with 10 μl 0.1N HCl + 90 μl NF-H2O) was added and reacted in a 37°C incubator for 10-13 minutes.

[0077] 1.5 Preparation of primer hybridization mixture and tissue hybridization

[0078] While the tissue sections are fixed, prepare the probe hybridization solution: 5 μM random probe (6N) and 5 μM splint. Add them to 5× SSC, mix thoroughly, and hybridize in a 55°C incubator for 10 minutes to form the primer hybridization mixture (Figure 2-①). Remove the sample and place it on ice. Add 5% (v / v) RNase inhibitor (RI). The random probe sequence is: 5'-CCTCCGACTGTGTGACTTAGACTTGCACTTGATGTGCTNNNNNN-3' (SEQ ID NO: 2); the splint sequence is: 5'-CAAGTGCAAGTCTAAGTCACACAGTCGGAGGCCAAGCGGTCTTAG-3' (SEQ ID NO: 3). After the reaction is complete, remove the chip, aspirate the surface liquid, and wash once with 190 μl of 5× SSC + 10 μl of RI. Add the hybridized primer hybridization mixture and hybridize for 30 minutes at room temperature. Then wash once with 5× SSC + RI.

[0079] 1.6 Reverse transcription (RT) and T4 ligase ligation

[0080] Aspirate the liquid on the chip surface and add a mixture of RT and T4 ligase (containing RT reagent, 3mM MgCl2, 10mM DTT, 1mM ATP, pH 8.3 @ 25°C; RT enzyme 2.5μl / 100μl and T4 ligase 5μl / 100μl). Place the chip in a 37°C incubator and react for 3 to 5 hours.

[0081] 1.7 Tissue Removal

[0082] After the reaction, remove the chip, blot the surface liquid, and rinse once with NF-H2O. Add tissue removal solution and place the chip in a 55°C incubator for 10 minutes. Remove the chip and aspirate the chip surface, then aspirate twice with NF-H2O to remove all tissue.

[0083] 1.8 cDNA recovery, amplification, and purification

[0084] 1.8.1 cDNA recovery: Add 400 μl / well cDNA release solution to the reaction wells of the above chip, cover the reaction wells with the chip with sealing film to seal, cover the plate cover and seal the outer ring to prevent volatilization, and react in a 55°C incubator for 3 hours to release cDNA; recover the liquid in the reaction wells into a new 1.5ml centrifuge tube, add 350 μl / well NF-H2O to wash the chip, and recover the washing solution into the same centrifuge tube to obtain the recovery solution. Add VAHTS to the 1.5ml centrifuge tube. TM DNA Clean Beads (VAZYME) were added to the recovery buffer at a ratio of 0.8:1, mixed by vortexing, and incubated at room temperature for 10 minutes. After a quick centrifugation, the tube was placed on a magnetic rack and allowed to stand for 3 minutes. Once the liquid had clarified, the supernatant was removed. 1 ml of freshly prepared 80% ethanol was added, mixed by vortexing, and then centrifuged briefly and allowed to stand for 30 seconds. The supernatant was discarded and the beads were washed once more with 80% ethanol. The beads were discarded and allowed to air dry at room temperature until the surface of the beads was free of reflections and cracks. 42 μl of NF-H2O was added for resolubilization. The beads were mixed by vortexing, and allowed to stand at room temperature for 5 minutes. After a quick centrifugation, the beads were allowed to stand on a magnetic rack for 3-5 minutes. Once the liquid had clarified, the supernatant was collected and transferred to a new PCR tube.

[0085] 1.8.2 cDNA amplification: Add 58 μl of PCR mixture (including 50 μl of cDNA HIFI Master Mix and 8 μl of cDNA Primers) to the above PCR tube, totaling 100 μl, and perform PCR reaction. The PCR reaction steps are: ① 95°C for 5 minutes, ② 98°C for 20 seconds, ③ 58°C for 20 seconds, ④ 72°C for 3 minutes, ② to ④ for 15 cycles, then 72°C for 5 minutes, remove and place on ice to obtain PCR product. Use Qubit dsDNA Mix to detect the concentration of cDNA after amplification: Invitrogen TMQubit dsDNA HS Buffer 198μl, Qubit dsDNA HS Reagent 200×1μl, cDNA product 1μl.

[0086] 1.8.3.cDNA purification: Transfer the PCR product from the previous step to a new 1.5ml centrifuge tube and equilibrate it with VAHTS at room temperature. TM DNA Clean Beads (VAZYME) were mixed in a volume ratio of 1:0.6, vortexed, and incubated at room temperature for 10 minutes. After a quick centrifugation, the tube was placed on a magnetic rack and allowed to stand for 3 minutes. After the liquid clarified, the supernatant was removed. 1 ml of freshly prepared 80% ethanol was added, the tube was allowed to stand for 30 seconds, and the supernatant was discarded. The beads were rinsed once more with 80% ethanol, the supernatant was discarded, and the beads were allowed to air dry at room temperature until the beads were glossy and cracked. 40 μl of NF-H₂O was added for resolubilization, vortexed, and allowed to stand at room temperature for 5 minutes. After a quick centrifugation, the tube was placed on a magnetic rack and allowed to stand for 3-5 minutes. After the liquid clarified, the supernatant was collected and transferred to a new 1.5 ml centrifuge tube to obtain the purified cDNA product. A 1 μl sample of cDNA was used to determine the concentration using the Qubit dsDNA HS Kit and the cDNA fragment distribution was analyzed using the Agilent 2100 High Sensitivity DNA Kit. (QC criteria: fragment sizes were primarily distributed between 1000 and 1500 bp.)

[0087] 1.9 cDNA shearing, amplification, and purification

[0088] 1.9.1 cDNA Shearing: Prepare the shearing reagents: 4 μl 5× TAG buffer, 1 μl shearing enzyme, and 20 ng of the purified cDNA product. Make up to 20 μl with NF-H2O. Incubate at 55°C for 10 minutes, then immediately place on ice to obtain the sheared product. Add 5 μl Stop buffer to the sheared product and incubate at room temperature for 5 minutes.

[0089] 1.9.2 Interrupt Product Amplification: Add 50 μl of Library HIFI Master Mix and 25 μl of Library PCR Primer Mix to the above reaction mixture, mix well, and then proceed with the PCR reaction. The PCR reaction steps are as follows: ① 95°C for 5 minutes, ② 98°C for 20 seconds, ③ 58°C for 20 seconds, and ④ 72°C for 30 seconds. Repeat ②-④ for 13 cycles, followed by 72°C for 5 minutes. Remove and place on ice to obtain the amplified product.

[0090] 1.9.3 Purification of amplified product: 100 μl of the above amplified product was added to VAHTS equilibrated at room temperature. TMAdd 60μl of DNA Clean Beads (VAZYME) to the supernatant, mix thoroughly with vortexing, incubate at room temperature for 5 minutes, centrifuge briefly, and place on a magnetic stand for 3 minutes. Once the liquid is clear, transfer the supernatant to a new PCR tube. Add 20μl of Vazyme Beads to the supernatant, mix thoroughly with vortexing, incubate at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand, and let it stand for 3-5 minutes until the liquid is clear. Discard the supernatant. Add 200μl of freshly prepared 80% ethanol, let it stand for 30 seconds, discard the supernatant, repeat the 80% ethanol wash, and let the beads dry at room temperature until the surface is free of reflections and cracks. Add 20μl of NF-H2O to dissolve the beads back. Mix thoroughly with vortexing, let it stand at room temperature for 5 minutes, centrifuge briefly, place on a magnetic stand, and let it stand for 3 minutes. Once the liquid is clear, transfer the supernatant to a new centrifuge tube to obtain the purified product. Take 1 μl of the purified product and measure the concentration using the Qubit dsDNA HS Kit. Then, use the Agilent 2100 High Sensitivity DNA Kit to check the fragment distribution. (QC criteria: fragment sizes are primarily distributed between 400 and 600 bp.)

[0091] 1.10 cDNA library circularization, sequencing, and data analysis

[0092] Transfer 40 ng of the purified product to a new PCR tube, add NF-H2O to a 20 μl volume, and add 20 μl of 2× Make DNB buffer (available with the MGISEQ-2000RS kit). Incubate the tube in a PCR instrument at 95°C for 3 minutes and then at 40°C for 3 minutes. The reaction mixture is then placed on ice. Then, 39 μl of RCA buffer, 1 μl of 10 mM ATP, and 4 μl of One Step Enzyme are added. The reaction is incubated at 30°C for 30 minutes. After removal, 20 μl of DNB stop buffer is added to create the circularized DNB library. The library is sequenced using the MGISEQ-2000RS sequencer. The data is automatically analyzed at https: / / uat.stomics.tech / sap / , resulting in a heatmap (Figure 6, left column).

[0093] Comparative Example 1: RT first, then T4 connection method

[0094] Steps 1.1 to 1.4 of Comparative Example 1 are the same as those of Example 1. Steps 1.5 and 1.6 of this comparative example are as follows:

[0095] 1.5 Random probe 6N hybridization with tissue and reverse transcription (RT)

[0096] 1.5.1 Hybridization of Random Probe 6N with Tissue: Dissolve 5 μM Random Probe 6N in 5× SSC, add 5% RI, and mix thoroughly. Wash the permeabilized tissue sections once with 5× SSC + RI. Then, add the mixed Random Probe 6N solution and hybridize at room temperature for 15 minutes. During this process, Random Probe 6N captures RNA in the tissue. Wash the sections once with 5× SSC + RI.

[0097] 1.5.2 Reverse transcription (RT) reaction: Add RT reaction solution (RT reagent, 3 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 8.3 at 25°C, 2.5 μl / 100 μl RT enzyme) to the chip surface and incubate at 42°C for 3 hours. Remove the chip and wash once with 0.1× SSC + RI.

[0098] 1.6 Splint hybridization and T4 ligation

[0099] 1.6.1 Splint hybridization: Dilute 5 μM splint in 5× SSC solution, add 5% volume of RI, mix well, apply to the washed sections in the previous step, and hybridize at room temperature for 15 minutes.

[0100] 1.6.2 T4 ligation: Add T4 ligation reaction solution (containing 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 at 25°C) and react at 16°C overnight.

[0101] Steps 1.7-1.10 of this comparative example are the same as steps 1.7-1.10 of Example 1. The principle diagram of the spatiotemporal transcriptome technology based on random probe RNA capture in this comparative example is shown in Figure 1. A comparison diagram of the operational flow charts of this comparative example (RT first, then T4 ligation) and Example 1 (RT+T4 ligation) is shown in Figure 3.

[0102] The results are shown in the right column of Figure 6. The specific analysis is as follows:

[0103] Mouse brain: The experiment used half a mouse brain. Using the method in Comparative Example 1, gene capture in this mouse brain was low. As shown in the figure, at a sequencing saturation of 17%, bin 200 only captured 206 gene types. In contrast, using the method in Example 1, at a sequencing saturation of 36%, bin 200 could already capture 2,920 genes. According to estimates, even at a saturation of 36%, bin 200 in Comparative Example 1 would only capture approximately 450 genes. Therefore, the method in Example 1 significantly increased gene capture efficiency in the mouse brain by 6.5 times. Sequencing saturation refers to the depth of sequencing a library. For example, if a library contains 10,000 gene sequences and a single lane is sequenced using the MGI-2000RS sequencer with a sequencing saturation of 17%, that lane is considered to have captured 17% of the genes, or 1,700 genes. The remaining undetected sequences can be captured by additional sequencing, i.e., by increasing the number of lanes sequenced. Bin 200 refers to the area enclosed by 200 DNBs x 200 DNBs on the capture chip. In this invention, the probes on the capture chip are generated using DNBs and, like DNBs, are arranged in a dot array with consistent row and pitch spacing. Bin 200 refers to the square area defined by 200 DNBs horizontally and vertically. When comparing technologies, a uniform bin size of 200 is used to compare the performance of different batches of experiments.

[0104] Mouse thymus: The test results of the mouse thymus are consistent with those of the mouse hemibrain. That is, when the method of comparative example 1 is used, when the sequencing saturation reaches 39%, there are 1900 types of genes detected under bin200; when the method of embodiment 1 is adopted, when the sequencing saturation is 41%, the types of genes detected by bin200 have reached 6260. The sequencing saturation of 41% and 39% is equivalent, and it can be considered that the sequencing depth is basically the same. Under this premise, the number of gene types captured by the method of embodiment 1 is 3.3 times that of the method of comparative example 1, and the improvement effect is more obvious. In addition, when the mouse thymus was tested using the method of comparative example 1, a diffusion phenomenon was found (the gray area around the tissue in the figure, the darker the area in the figure, the less captured, and the whiter the area, the higher the captured amount). When the method of embodiment 1 was used, the diffusion phenomenon was significantly weakened, which is an additional discovery.

[0105] Mouse spleen: The test results for the mouse spleen were consistent with those for the mouse hemibrain and mouse thymus. Using the method of Comparative Example 1, at a sequencing saturation of 81%, bin200 captured 3,010 gene types. An estimated number of bin200 gene types at a sequencing saturation of approximately 40% is approximately 1,500. However, using the method of Example 1, at a sequencing saturation of 42%, bin200 captured 6,340 gene types, significantly increasing gene capture efficiency by 4.2 times.

[0106] Comparative Example 2: T4 connection first, then RT method

[0107] First, splint is added to hybridize with the oligonucleotide strands immobilized on the chip (Figure 4-1). Tissue sections are then mounted, fixed, and permeabilized, followed by the addition of random probe 6N to capture RNA (Figure 4-2). Next, T4 ligase is added to ligate the immobilized oligonucleotide sequence of random probe 6N to the oligonucleotide sequence on the chip, connecting the RNA-captured random probe 6N and the oligonucleotide strands on the chip into a single strand (Figure 4-3). RT enzyme is then added to reverse transcribe cDNA (Figure 4-4). Testing revealed that this method yielded low capture yields, as shown in the left panel of Figure 7. Figure 7 uses mouse brain as experimental material. Using the T4 ligation followed by RT method, splint is first ligated to the immobilized oligonucleotide sequence on the chip, followed by RNA capture and reverse transcription using random probes, and then T4 ligase is added for ligation. The cDNA library obtained in this way was sequenced by MGI-2000RS. When the sequencing saturation was 59%, the number of gene types captured by bin200 was 1752. However, when the sequencing saturation was about 59%, the number of gene types captured by bin200 using the method in Example 1 could reach about 4800, and the capture efficiency was increased by 2.7 times.

[0108] Using a variable temperature hybridization method, the splint is first hybridized with the fixed oligonucleotide sequence of the random probe at a high temperature (55°C). Since the hybridization distance between the splint and the fixed oligonucleotide sequence on the chip is relatively short, this section will melt at high temperature, ensuring that the splint fully hybridizes with the random probe at high temperature. The temperature is then restored to a low temperature (around 25°C) to allow the splint to hybridize smoothly with the fixed oligonucleotide sequence on the chip. The cDNA library obtained in this way was sequenced by the MGI-2000RS. At a sequencing saturation of 58%, the number of gene types captured by bin200 was 2940. Compared with the method in Example 1, at a sequencing saturation of approximately 59%, the number of gene types captured by bin200 reached approximately 4800, a 1.6-fold increase in capture efficiency.

[0109] The specific steps are:

[0110] The steps 2.1-2.2 of this comparative example are the same as steps 1.1-1.2 of Example 1. Steps 2.3-2.6 of this comparative example are as follows:

[0111] 2.3 Chip processing and tissue patching

[0112] 2.3.1 Hybridization of splint with the capture chip: Carefully pick up the capture chip with tweezers and place it in a new 24-well plate. Dissolve 5 μM splint in 5× SSC, mix thoroughly, and coat the capture chip surface. Hybridize at room temperature for 15 minutes. The splint will hybridize with the immobilized oligonucleotide sequence on the capture chip surface, as shown in Figure 4-①.

[0113] 2.3.2 Tissue patch: Wash the chip twice with 400 μl of 0.1× SSC, rinse thoroughly with nuclease-free water (NF-H2O), and bake the chip at 37°C to dry. Cut frozen OCT tissue into 10 μm sections, flatten the sections, and hold the capture chip with forceps. Quickly approach the frozen section, and the tissue section will automatically adhere to the chip. Then bake the attached tissue chip at 37°C for 3-5 minutes.

[0114] 2.4 Tissue section fixation and permeabilization

[0115] The baked chip was placed in a -20°C pre-cooled methanol solution for 30 minutes, and then taken out to dry the methanol. Permeabilization reagent (permeabilization enzyme mixed with 10 μl 0.1N HCl + 90 μl NF-H2O) was added and reacted in a 37°C incubator for 10-13 minutes.

[0116] 2.5 Random Probe 6N Hybridization and T4 Ligation

[0117] 2.5.1 Random Probe 6N Hybridization: Dissolve 5 μM random probe 6N in 5× SSC, add 5% RI, and mix thoroughly. Apply the random probe solution to the chip and hybridize for 15 minutes at room temperature. At this point, random probe 6N will hybridize with and capture RNA from the tissue section (Figure 4-②). Wash once with 5× SSC + RI.

[0118] 2.5.2 T4 Ligation: Add T4 ligation reaction solution (10mM MgCl2, 1mM ATP, 10mM DTT, pH 7.5 @ 25°C) to the cleaned chip surface and incubate at 16°C overnight. Theoretically, the random probe 6N that captured the RNA will ligate to the oligonucleotide sequences on the chip (Figure 4-③). After the reaction, wash once with 0.1× SSC + RI.

[0119] 2.6 Reverse transcription (RT)

[0120] Add RT reaction solution (RT reagent, 3 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 8.3 at 25°C, 2.5 μl / 100 μl RT enzyme) to the chip and react at 42°C for 3 hours. Wash once with 0.1× SSC + RI.

[0121] Steps 2.7 to 2.10 of this comparative example are the same as steps 1.7 to 1.10 of Example 1.

[0122] Comparative Example 3: Variable Temperature Hybridization Method

[0123] After random probe 6N is added to capture RNA and reverse transcribe it into cDNA, splint is added and hybridized with the fixed oligonucleotide sequence on random probe 6N at 55°C. Then, the splint is placed at room temperature to hybridize with the oligonucleotide chain fixed on the chip.

[0124] When designing the splint, the hybridization length with the fixed oligonucleotide sequence of random probe 6N was set to 31 bases (Tm value 65°C), while the hybridization length with the oligonucleotide chain on the chip was set to 14 bases (Tm value 45°C). This allows for hybridization using a variable temperature hybridization method. After adding the splint, the splint is first hybridized to the fixed oligonucleotide sequence of random probe 6N at a high temperature (55°C). Because the hybridization length between the splint and the nucleic acid chain on the chip is short, even hybridization at 55°C will melt due to the high temperature. This method prevents the splint from hybridizing with the nucleic acid chain on the chip alone, which would render the nucleic acid chain ineffective. After the splint hybridizes with the fixed oligonucleotide sequence of random probe 6N, it is washed once with 5× SSC preheated at 55°C to remove excess splint. Then, 5× SSC is added to return to room temperature to allow the splint hybridized with random probe 6N to hybridize with the nucleic acid chain on the chip. Then, T4 ligation reaction solution is added to initiate the ligation reaction. However, after testing, the results are shown in the right figure of Figure 7, and it was found that the capture amount of this method was not ideal, and the capture was extremely uneven.

[0125] Steps 3.1-3.4 of this comparative example are the same as steps 1.1-1.4 of Example 1. Steps 3.5 and 3.6 of this comparative example are as follows:

[0126] 3.5 Random Probe 6N Hybridization with Tissue and Reverse Transcription (RT)

[0127] 3.5.1 Hybridization of Random Probe 6N with Tissue: Dissolve 5 μM Random Probe 6N in 5× SSC, add 5% RI, and mix thoroughly. Wash the permeabilized tissue sections once with 5× SSC + RI. Then, add the mixed Random Probe 6N solution and hybridize at room temperature for 15 minutes. During this process, Random Probe 6N captures RNA in the tissue. Wash the sections once with 5× SSC + RI.

[0128] 3.5.2 Reverse Transcription (RT) Reaction: Add RT reaction solution (RT reagent, 3 mM MgCl2, 10 mM DTT, 1 mM ATP, pH 8.3 at 25°C, 2.5 μl / 100 μl RT enzyme) to the chip surface and incubate at 42°C for 3 hours. Wash once with 0.1× SSC + RI.

[0129] 3.6 Splint hybridization and T4 ligation

[0130] 3.6.1 Splint hybridization: Dilute 5 μM splint in 5× SSC solution, add 5% volume of RI, mix well, apply to the washed sections in the previous step, and hybridize at 55°C for 15 minutes. Remove the sections and wash once with 5× SSC + RI solution preheated at 55°C, then add 5× SSC solution, return to room temperature, and incubate for 15 minutes.

[0131] 3.6.2 T4 ligation: Add T4 ligation reaction solution (containing 10 mM MgCl2, 1 mM ATP, 10 mM DTT, pH 7.5 @ 25°C) and react at 16°C overnight.

[0132] Steps 3.7-3.10 of this comparative example are the same as steps 1.7-1.10 of Example 1.

[0133] Comparative Example 4: Splint and random probes are used separately

[0134] In Example 1 of the present invention, after combining the RT and T4 ligation steps, random probe 6N and splint were hybridized at a molar ratio of 1:1 to form a mixture (① in FIG. 2 ), and then RNA capture was performed.

[0135] This comparative example attempted to introduce splint and random probe 6N separately, potentially allowing hybridization between the splint and the oligonucleotide chains on the chip (see ① in Figure 5 ). Subsequently, when the RT and T4 ligation reaction system was added, a portion of the oligonucleotide chains immobilized on the chip polymerized upward using the splint as a template, forming an extended sequence (see ② in Figure 5 ). The remaining steps were the same as in Example 1.

[0136] However, it was found that this would cause the sequence of the oligonucleotide chains on the chip to change. The fixed oligonucleotide sequence of the random probe 6N could no longer hybridize with the extended sequence through splint, resulting in the oligonucleotide chains on the chip being scrapped and unable to capture the RNA in the sample.

[0137] Therefore, hybridizing the random probe 6N and splint to form a mixture first can minimize the probability of hybridization of the individual splints with the oligonucleotide chains on the chip, thereby maintaining the original hybridization function of the oligonucleotide chains on the chip.

[0138] Although the above describes specific embodiments of the present invention, it should be understood by those skilled in the art that these are merely illustrative and that various changes or modifications may be made to these embodiments without departing from the principles and essence of the present invention. Therefore, the scope of protection of the present invention is defined by the appended claims.

Claims

1. A method for capturing nucleic acid, characterized in that: It includes the following steps: The random probe and the oligonucleotide chain fixed on the capture chip are hybridized by splint oligonucleotides, the RNA in the sample captured by the random probe is reverse transcribed into cDNA, and the cDNA is connected to the oligonucleotide chain, and the reverse transcription and the connection are carried out in the same reaction system.

2. The method according to claim 1, wherein It includes the following steps: (1) hybridizing the random probe and the splint sequence; wherein the 5' end of the random probe is partially complementary to the 5' end of the splint sequence; (2) the random probe hybridizes with RNA in the sample; wherein the 3' end of the random probe is partially complementary to the 3' end of the RNA; (3) hybridizing the splint sequence with an immobilized oligonucleotide sequence; preferably, the oligonucleotide sequence is immobilized on a capture chip; (4) In the same reaction system, cDNA is synthesized using the RNA in the complex formed by the simultaneous hybridization in (1), (2) and (3) as a template, and the fixed oligonucleotide sequence and the random probe on the complex are connected, so that the fixed oligonucleotide, the random probe and the cDNA form a long single-stranded nucleotide containing cDNA in the 5' to 3' direction.

3. The method according to claim 2, characterized in that In step (1), the random probe includes 6-20N; In step (3), the capture chip is placed at 30-45° C., for example, 37° C., for reaction for 3-24 hours, for example, 3-5 hours; In the reaction system of step (4), reverse transcriptase and DNA ligase are present simultaneously; Preferably, the sample comprises a tissue sample.

4. The method according to claim 1, wherein The steps further include: (5) The liquid on the surface of the capture chip is aspirated, and a mixture of reverse transcriptase and T4 ligase 1 is added. The capture chip is placed in an incubator and reacted for 3-24 hours.

5. The method according to claim 4, wherein The mixed solution 1 contains reverse transcription reagent, 3-25mM MgCl2, 1-50mM DTT, and ≥1mM ATP; The volume ratio of the reverse transcriptase to the T4 ligase is 2:1 or 1:1; and / or, The temperature of the incubator is 35-42°C.

6. The method according to claim 4, wherein The pH of the mixed solution is 8.

3.

7. The method according to claim 4, wherein Before step (5), the method further includes: (i) While the tissue section is being fixed to the capture chip, a probe hybridization solution is prepared, mixed with 5×SSC, and hybridized in an incubator at 50-60° C., for example, 55° C., for 5-30 minutes, for example, 10 minutes, to form a primer hybridization mixture. The mixture is then removed from the incubator, cooled, and 2-15% by volume, for example, 5% by volume, of an RNase inhibitor is added; (ii) removing the capture chip, removing the surface liquid, washing the capture chip with mixed solution 2, adding the primer hybridization mixture, hybridizing at room temperature for 15 to 60 minutes, and then washing again with mixed solution 2; The mixed solution 2 is 5×SSC and RNase inhibitor; The probe hybridization solution contains random probes and splint.

8. The method according to claim 5, wherein The tissue sections are sections of frozen OCT tissue; The probe hybridization solution is a random probe and splint with a molar ratio of 1:1; The cooling method is to place the mixture on ice and / or place the mixture at room temperature until the mixture cools to room temperature, and / or, The volume ratio of 5×SSC to RNase inhibitor in the mixed solution 2 is (5-30):1, for example, 19:

1.

9. The method according to claim 7 or 8, wherein The sequence of the random probe is shown in SEQ ID NO: 2; the sequence of the splint is shown in SEQ ID NO:

3.

10. The method according to claim 1, wherein The method further comprises the following steps: (a) after completing step (ii), removing the sample tissue on the capture chip; (b) recovering cDNA from the capture chip; optionally, further comprising steps of cDNA amplification and purification.

11. The method according to any one of claims 1 to 10, characterized in that The capture chip includes a capture probe, wherein the capture probe includes spatial barcode position information and a fixed oligonucleotide sequence; the fixed oligonucleotide sequence hybridizes with the 3' end of the splint.

12. The method according to claim 11, characterized in that The capture probe is 5'Nm-SEQ ID NO: 1-3', wherein m is 8-30.

13. The method according to claim 12, characterized in that Said m≥25.

14. A high-throughput sequencing method, characterized in that: It includes the following steps: (I) obtaining the long single-stranded nucleotide sequence containing cDNA to be sequenced according to the method of any one of claims 1 to 13; (II) Construction of cDNA library; (III) Perform sequencing and analyze the obtained data.

15. The method according to claim 14, wherein (I) further includes the steps of cDNA shearing, amplification and purification; and / or, in (II), the cDNA library is circularized to obtain a circularized DNB library.

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