Spatial transcriptome in-situ sequencing method for tissue slices

Abstract

A space transcriptome in-situ sequencing method for tissue slices relates to the field of gene sequencing, and comprises the following steps: 1) capturing mRNA in situ; 2) carrying out reverse transcription to obtain cDNA; (3) removing tissues and retaining cDNA (complementary deoxyribonucleic acid); 4) carrying out cDNA probe hybridization; 5) performing in-situ amplification; 6) decoding and sequencing the fluorescent probe; and 7) cleaning the probe, and sequencing for multiple rounds. According to the invention, RNA is captured in situ and is reversely transcribed into cDNA, nucleic acid imprinting transfer is realized, background interference is removed, the problem of RNA degradation is solved, and multiple rounds of hybridization are allowed to increase the detection flux. The nucleic acid sequence is covalently bound with a slide to prevent a signal point from shifting and falling off. A DNA coding padlock probe is used for identifying genes, amplifying and amplifying signals, and gene expression is revealed through sequencing and decoding. After the probe is cleaned, the probe is replaced for hybridization amplification, so that the flux is improved, and space crowding and fluorescence signal crowding are reduced.

Description

Technical Field

[0001] The present invention relates to the field of gene sequencing, and in particular to a method for in situ sequencing of spatial transcriptomes of tissue sections. Background Art

[0002] The single-cell isolation step in the single-cell transcriptome sequencing process requires tissue dissociation, which results in the loss of information about the spatial location of cells in the tissue. Spatial transcriptome technology is a technology that combines imaging, biomarkers, sequencing, and bioinformatics tools to spatially locate gene expression in tissue sections. It can reveal the spatial distribution of each cell type in the tissue, the interaction between each cell population, and map the gene expression of different tissue regions. It has far-reaching application value in understanding the mechanisms of disease and cancer.

[0003] Existing spatial transcriptome technologies mainly include microdissection technology, in situ capture technology, in situ hybridization technology, and in situ sequencing technology.

[0004] In situ hybridization and in situ sequencing techniques can determine the spatial location of RNA molecules at the subcellular level with high resolution. However, due to the limitations of the instrument and the number of fluorescent channels, the method has a low throughput; and because RNA is easily degraded, the method has a long process and is prone to signal point loss; at the same time, this method is based on probe hybridization to locate RNA, so it can only detect known RNA sequences.

[0005] In situ capture technologies such as ST-seq and slide-seq can sequence RNA at a high throughput, but their resolution often does not reach the single-cell level, and they have limitations such as low capture rate and gaps between capture points. At the same time, this method locates cells based on spatial encoding, but cannot determine the spatial position of RNA within the cell.

[0006] Existing sequencing methods capture RNA in tissues in situ and perform imaging (such as EEL FISH). Removing tissue can reduce z-axis imaging time, while reducing background and steric hindrance of hybridization. However, sequencing is still based on RNA hybridization, and the above problems have not been well solved. Summary of the invention

[0007] The purpose of the present invention is to solve the above problems in the prior art and provide a method for in situ sequencing of spatial transcriptome of tissue sections.

[0008] In order to achieve the above object, the present invention adopts the following technical scheme:

[0009] A method for in situ sequencing of spatial transcriptomes of tissue sections, comprising the following steps:

[0010] 1) In situ capture of mRNA;

[0011] 2) Reverse transcription of mRNA to obtain cDNA;

[0012] 3) Remove the tissue and keep the cDNA in place;

[0013] 4) Probe hybridization of the single-stranded cDNA in situ;

[0014] 5) Perform in situ amplification;

[0015] 6) Decoding by fluorescent probes enables in situ sequencing of RNA in tissues;

[0016] 7) Wash the hybridization probe, leaving the cDNA single strand in step 3);

[0017] 8) Repeat steps 4) to 6) to achieve multiple rounds of RNA in situ sequencing.

[0018] In step 1), primers are first modified on a glass slide, and then a tissue section is attached to the region modified with the primers and mRNA is captured.

[0019] In step 1), mRNA capture is covalently bonded to the surface of the glass slide, or covalently bonded to the hydrogel mesh structure fixed on the surface of the glass slide. Covalent bonding methods include but are not limited to covalent bonding of glass slides modified with surface aldehyde groups or carboxyl groups, epoxy groups, amino groups, NHS groups, etc., and amino-modified DNA.

[0020] In step 1), the in situ capture of mRNA includes but is not limited to the following methods: digestion with pepsin to release mRNA in the tissue for in situ capture; destroying the membrane structure with surfactants to release mRNA in the tissue for in situ capture; using an electric field to direct the mRNA to the surface of a glass slide for capture; using centrifugation to direct the mRNA to the surface of a glass slide for capture.

[0021] In step 2), the reverse transcriptases include but are not limited to the following: AMV reverse transcriptase, MMLV reverse transcriptase, PDB:1HMV reverse transcriptase.

[0022] In step 3), the method of removing tissue includes but is not limited to the following methods: using proteinase K to digest proteins in the tissue and removing the tissue by washing with a solution; using trypsin to digest proteins in the tissue and removing the tissue by washing with a solution; using collagenase to digest proteins in the tissue and removing the tissue by washing with a solution.

[0023] In step 4), a primer probe targeting the cDNA after reverse transcription of the target mRNA is added for hybridization.

[0024] In step 4), the primer probes targeting different cDNAs contain different coding sequences.

[0025] In the present invention, the amplification method and the fluorescent probe hybridization method include the following methods: the hybridization probe is circularized, RCA amplification is performed, and then a fluorescent probe is used for hybridization, and detection is performed by imaging; amplification is performed by the HCR method, and the probe has a fluorescent group, and detection is performed by imaging; amplification is performed by the LAMP method, and then a fluorescent probe is used for hybridization, and detection is performed by imaging.

[0026] In step 6), sequencing probes with different fluorophores are used for hybridization, and then the results of multiple rounds of hybridization and imaging are decoded to obtain the spatial distribution information of mRNA.

[0027] Hybridization probe cleaning methods include but are not limited to the following: introducing restriction endonuclease action sites into the amplified product, using restriction endonuclease to degrade it into short fragments and clean it; introducing U bases into the amplified product, using User enzyme to degrade it into short fragments and clean it; introducing CRISPR gene sequences into the amplified product, using Cas protein to degrade it into short fragments and clean it.

[0028] Compared with the prior art, the technical solution of the present invention has the following beneficial effects:

[0029] At present, the in situ sequencing method based on RNA probe hybridization imaging has the following problems: 1. RNA is easily degraded, which will cause the loss of signal points; 2. The hybridization steric hindrance in the tissue is large and the hybridization time is long; 3. The steric hindrance in the amplification process causes the amplification reaction efficiency to be low; 4. The tissue will cause certain background interference during the imaging process; 5. The imaging is limited by the microscope resolution, and there is a problem of crowded fluorescent signals. In order to solve the above problems, the present invention first captures RNA in situ by primers fixed on the glass slide, and reversely transcribes it to convert it into cDNA that is not easy to degrade, so that the sequence in the subsequent steps is more stable. And because the cDNA is fixed on the surface of the glass slide, the position will be relatively fixed during multiple rounds of imaging. After reverse transcription, proteinase K is used to remove the tissue to reduce steric hindrance and background interference in imaging. After treatment with an alkaline solution, the single-stranded cDNA on the surface of the glass slide is probe hybridized, and then in situ amplified, and then the RNA in situ sequencing in the tissue is realized by hybridization of the fluorescent probe. After the end of this round of in situ sequencing, the RCA amplification product is cleaned, and a hybridization probe targeting other cDNA is added, and then in situ amplification is performed, and hybridization detection by the fluorescent probe is performed. This step effectively reduces spatial crowding during the amplification reaction and solves the problem of fluorescent signal crowding in imaging, further improving detection throughput. BRIEF DESCRIPTION OF THE DRAWINGS

[0030] Figure 1Characterize the slide modification results.

[0031] Figure 2 The olfactory bulb tissue was permeabilized for 10 min (left), 15 min (middle), and 20 min (right) before reverse transcription, and the reverse transcription results were characterized by fluorescence.

[0032] Figure 3 It is the result of RCA, detected by in situ hybridization with fluorescent probe.

[0033] Figure 4 Electron microscopy characterization of RCA products.

[0034] Figure 5 To characterize the displacement and shedding of fluorescent signal points, they are the fluorescence images of 10 rounds of hybridization (a) and the statistics of fluorescent signal points of 10 rounds (b).

[0035] Figure 6 Four rounds of fluorescence characterization images of 16 genes in olfactory bulb tissue.

[0036] Figure 7 Fluorescence image decoding results of 16 gene sequencing in olfactory bulb tissue.

[0037] Figure 8 The RCA amplification products were characterized by fluorescence microscopy before washing (first from left), after washing (second from left), after hybridization with the same probe and RCA amplification (second from right), and after the two RCA amplification images were overlapped (first from right).

[0038] Fig. 9 These are the imaging results of three times of simultaneously targeting Slc17a7 and Cck (left) and separately targeting Slc17a7 (middle) and Cck (right) in the same olfactory bulb tissue and performing RCA amplification. DETAILED DESCRIPTION

[0039] In order to make the technical problems, technical solutions and beneficial effects to be solved by the present invention clearer and more understandable, the present invention is further described in detail below in conjunction with the accompanying drawings and embodiments.

[0040] Example 1

[0041] The following takes the olfactory bulb tissue as an example.

[0042] 1. Slide modification

[0043] 1. Prepare monomer solution: 40% acrylamide 10μL, 2% methylene bisacrylamide 10μL, 100μM acrylamide-polyT 5μL, 1×TE 64.8μL;

[0044] 2. Prepare APS solution (2%, freshly prepared and used);

[0045] 3. Add 0.2 μL TEMED to the monomer solution, then add 10 μL APS solution, mix well, and drop it onto the surface of the glass slide wiped with affinity silane;

[0046] 4. Cover the glass slide with plastic film to flatten the droplets, pad both sides with lens cleaning paper, and place in an oven at 40°C for 30 minutes;

[0047] 5. Place the slide in ultrapure water, peel off the plastic film on the surface, and wipe off the excess gel;

[0048] 6. Gel dehydration: Place the slides in 70%, 85%, and 99% ethanol for 1 min each, air-dry the surface, and store at 4°C.

[0049] 2. mRNA Capture

[0050] 1. Stick the frozen sections of the olfactory bulb to the modified slide area and store at -80℃;

[0051] 2. The frozen sections of the olfactory bulb were placed in a 37°C oven for 1 min and then fixed with methanol at -20°C for 30 min;

[0052] 3. Add isopropanol from the side of the tissue until it covers the tissue, incubate at room temperature for 1 min, tilt the slide to remove excess isopropanol on the surface, and air-dry the tissue;

[0053] 4. Use a tissue pen to draw a circle with a diameter of about 5 mm in the slice area;

[0054] 5. Prepare permeabilization solution (0.1% pepsin hydrochloric acid solution (pH = 2)) and equilibrate at 37°C for 15 minutes;

[0055] 6. Add 50 µL of pepsin solution into the hydrophobic circle, evenly cover the tissue without creating bubbles, tap the slide to ensure even coverage, and place in a 37°C oven for permeabilization for 15 minutes (the incubation time is determined by tissue type, slice thickness, etc.);

[0056] 7. Aspirate the permeabilization solution and wash twice with 0.1×SSC.

[0057] 3. Reverse Transcription

[0058] 1. Wash once with 1×RT buffer;

[0059] 2. Prepare RT Mix on ice and mix thoroughly by pipetting. The formula of RT Mix is ​​as follows: 21 µL dd H 2 O, 8µL RTBuffer (5×), 4µL dNTPs (10mM), 1µL RNase Inhibitor (40U / µl), 2µL RT enzyme (200U / µl);

[0060] 3. Add the above RT mix into the hydrophobic circle and incubate at 42°C for 2 h.

[0061] 4. Tissue Digestion

[0062] 1. Remove the RT mix and wash with TE-SDS, TE-TW, and Tris-HCl (10 mM, pH=8.0) respectively;

[0063] 2. Prepare tissue digestion solution at room temperature and mix well by pipetting; the formula of tissue digestion solution is as follows: 26.4µL Tris-HCl (10mM), 4µL proteinase K (20mg / mL), 1.6µL NaCl (5M), 4µL EDTA (0.5M), 4µL SDS (20%);

[0064] 3. Add tissue digestion solution and incubate at 55°C for 3 hours;

[0065] 4. After the tissue digestion is completed, remove the digestion solution, place the slides in 2×SSC 0.1%SDS and wash them up and down 15 times, shake them vigorously, and then wash the slides with 0.1×SSC.

[0066] 5. Single Chain

[0067] 1. Prepare 50µL KOH (0.08M), add it into the hydrophobic circle, and react at room temperature for 5 minutes;

[0068] 2. Remove most of the KOH and wash with 100 µL Tris-HCl (10 mM, pH = 8.0), TE-TW, and Tris-HCl (10 mM, pH = 8.0) respectively.

[0069] VI. Padlock probe hybridization

[0070] 1. Wash 3 times with 50 μL DEPC-PBST;

[0071] 2. Prepare Padlock hybridization reagent: 32 μL DEPC H 2 O, 15μL SSC (20×), 0.5μL Tween20 (1%), 2.5μL padlock (2μM);

[0072] 3. Add Padlock hybridization reagent and incubate at 37°C for 1 h;

[0073] 4. Wash three times with 50 μL DEPC-PBST;

[0074] 6. Add 50 μL 1×Hyb buffer 2 (2× SSC, 20% forfamide), incubate at 37°C for 5 min, repeat 3 times;

[0075] 7. Wash three times with 50 μL DEPC-PBST.

[0076] 7. Probe Ringing

[0077] 1. Prepare 50 μL ligation reaction solution: 42.5 μL DEPC H 2 O, 5μL T4 DNA ligase buffer (10×), 2.5μL T4 DNA ligase (400 U / μL);

[0078] 2. Add reaction solution and incubate at 37℃ for 0.5 h;

[0079] 3. Wash three times with 50 μL DEPC-PBST.

[0080] 8. Side primer hybridization

[0081] 1. Prepare Side Primer hybridization reaction solution: 35 μL DEPC H 2 O, 2.5 μL SSC (20×), 12.5 μL Sideprimer (2 μM);

[0082] 2. Add reaction solution and incubate at 37℃ for 0.5 h;

[0083] 3. Wash three times with 50 μL DEPC-PBST.

[0084] In this embodiment, the RCA method is used to amplify the probe in situ, and then the ISS probe is used for detection.

[0085] 9. RCA Amplification

[0086] 1. Prepare 50 μL RCA reagent: 30 μL DEPC H 2 O, 5 μL Phi29 DNA polymerase (10 U / μL), 5 μL Phi29 buffer (10×), 5 μL dNTP (10 mM), 5 μL Glycerol (50%);

[0087] 2. Add RCA reagent and incubate at 30°C overnight;

[0088] 3. Wash three times with 50 μL DEPC-PBST.

[0089] 10. ISS Response

[0090] 1) Prepare ISS mixes of Base 1 to Base 4 respectively, and then perform the following operations; the formula of ISS mix is ​​as follows: 32 μL DEPC H 2 O, 0.5μL *4 kinds of Fluorescent probe (10 μM), 5μL Anchor (10 μM), 1μL T4 DNA ligase (5 U / μL), 5μL T4 DNA ligase buffer (10×);

[0091] 2) Add 50 μL of ISS mix reagent and incubate at 30°C for 45 min;

[0092] 3) Wash three times with 50 μL DEPC-PBST;

[0093] 4) Add 20 μL of 50% glycerol to seal the slide and use a fluorescence microscope to image;

[0094] 5) Soak the slides in DEPC-PBST and remove the coverslip;

[0095] 6) Wash three times with 50 μL DEPC-PBST;

[0096] 7) Add 50 μL formamide and incubate at 50°C for 10 min;

[0097] 8) Wash three times with 50 μL DEPC-PBST and observe each channel under a microscope to see if there is any residual signal under normal exposure.

[0098] 11. RCA product cleaning

[0099] 1. Prepare 50 μL of endonuclease probe hybridization solution: 22.5 μL DEPC H 2 O, 2.5 μL probe (10 μM), 25 μL 2×Hyb buffer2 (4× SSC, 40% formamide);

[0100] 2. Add 50 μL of reagent and incubate at room temperature for 15 min;

[0101] 3. Wash three times with 50 μL DEPC-PBST;

[0102] 4. Prepare 50 μL restriction enzyme solution: 42.5 μL DEPC H 2 O, 2.5μL SgrDI (5 U / μL), 5μL Buffer R (10×);

[0103] 5. Add 50 μL of reagent and incubate at 37°C for 1 h;

[0104] 6. Wash three times with 50 μL DEPC-PBST.

[0105] 12. Multiple rounds of sequencing

[0106] Repeat steps 5 to 10 to perform a new round of sequencing analysis.

[0107] 13. Results and Analysis

[0108] The results of slide modification are shown in Figure 1 The modified primers were hybridized using fluorescent poly(A) chains, and the modification results were characterized by fluorescence microscopy. The brightness of the modified slides was uniform and the signal-to-background ratio was high.

[0109] Permeabilization time and reverse transcription characterization are shown in Figure 2 The olfactory bulb tissue was permeabilized for 10 minutes, 15 minutes, and 20 minutes before reverse transcription, and the reverse transcription results were characterized by fluorescence. The fluorescence images show that this method successfully performed reverse transcription on the glass slide, and the olfactory bulb tissue permeabilized for 15 minutes had a clear and complete outline and a high signal-to-background ratio.

[0110] RCA results are shown in Figure 3 and Figure 4 The single-stranded cDNA was probe hybridized and RCA amplified, and the RCA product was characterized by a microscopic imaging system and a cryo-electron microscope. The microscopic imaging system characterized that the tissue outline was obvious and clear, and the RCA amplification product was obtained by cryo-electron microscopy.

[0111] Fluorescence signal point displacement and shedding characterization are shown in Figure 5 , the fluorescence intensity slightly weakened after multiple rounds of washing and imaging. The number of fluorescent signal points in the same field of view was counted. During multiple rounds of imaging, the number of fluorescent signal points in the same field of view remained basically unchanged, indicating that the RCA product was not displaced or detached during the washing process.

[0112] Multiple rounds of imaging and fluorescence signal decoding are shown in Figure 6 and Figure 7 , 16 genes in the olfactory bulb tissue were encoded, hybridized with the encoded probes, and amplified by RCA, followed by in situ sequencing with sequencing probes. Fluorescent images of multiple rounds of hybridization were decoded to obtain the distribution map of these 16 genes in the olfactory bulb tissue.

[0113] RCA product cleaning results are shown in Figure 8After RCA amplification, a clear fluorescent signal was obtained by fluorescence microscopy. After restriction endonuclease treatment, the fluorescent signal disappeared, indicating that the amplified product was cleaned. The same sample was hybridized again with the same probe and subjected to RCA amplification reaction. The product of the re-amplification was characterized by fluorescence microscopy, and a clear fluorescent signal was obtained. The images of the two amplification characterizations can overlap well, indicating that multiple RCA amplifications were successfully performed on the same sample.

[0114] The effect of RCA product cleaning is shown in Fig. 9 , firstly, probes targeting Slc17a7 and Cck in the mouse olfactory bulb were added, and RCA amplification was performed. After imaging, the amplified products were cleaned with restriction endonucleases. The probe targeting the Slc17a7 gene was re-added, and amplification was performed. After imaging, the amplified products were cleaned again. The probe targeting the Cck gene was added, and imaging was performed after amplification. The results showed that the problem of fluorescence signal crowding was effectively solved by cleaning the RCA amplified products with restriction endonucleases (indicated by the white arrows). In addition, the number of signal points obtained by detecting the two genes separately was greater than the number of signal points obtained by detecting the two genes simultaneously, indicating that RCA product cleaning can also reduce the problem of low amplification efficiency caused by crowding during the amplification process.

[0115] The present invention captures RNA in tissue in situ and reverse transcribes it into cDNA, realizes nucleic acid imprint transfer in tissue, removes background interference at the same time, effectively solves the problem of RNA degradation and instability in the in situ sequencing method, and allows multiple rounds of hybridization detection, thereby increasing the detection throughput. The nucleic acid sequence is covalently bound to the surface of the glass slide, which can effectively solve the problem of signal point displacement and shedding in imaging. The target gene is identified by a DNA-encoded padlock probe, and the signal is amplified by amplification. The DNA coding sequence is then decoded by sequencing to reveal the situation of gene expression in space. Finally, the hybridization probe is cleaned, and single-stranded DNA hybridization is performed using probes targeting other genes, and amplification detection is performed to further improve the throughput of the method, effectively reduce spatial crowding during the reaction process, and solve the problem of fluorescent signal crowding during imaging.

[0116] The above description is only a preferred embodiment of the present invention, and therefore cannot be used to limit the scope of the present invention. That is, equivalent changes and modifications made according to the patent scope of the present invention and the contents of the specification should still fall within the scope of the present invention.

Claims

1. A method for in situ sequencing of spatial transcriptome of tissue sections, characterized in that: The following steps are involved: 1) In situ capture of mRNA; 2) Reverse transcription of mRNA to obtain cDNA; 3) Remove the tissue and keep the cDNA in place; 4) Probe hybridization of the single-stranded cDNA in situ; 5) Perform in situ amplification; 6) Decoding by fluorescent probes enables in situ sequencing of RNA in tissues; 7) Wash the hybridization probe, leaving the cDNA single strand in step 3); 8) Repeat steps 4) to 6) to achieve multiple rounds of RNA in situ sequencing.

2. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: In step 1), primers are first modified on a glass slide, and then a tissue section is attached to the region modified with the primers and mRNA is captured.

3. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 2, characterized in that: In step 1), mRNA capture is bound to the surface of the glass slide via covalent bonds, or is covalently linked to a hydrogel mesh structure fixed on the surface of the glass slide.

4. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: In step 1), the in situ capture of mRNA includes the following methods: digestion with pepsin to release mRNA in the tissue for in situ capture; destroying the membrane structure with surfactants to release mRNA in the tissue for in situ capture; using electric field to direct the mRNA to the surface of the glass slide for capture; using centrifugation to direct the mRNA to the surface of the glass slide for capture.

5. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: In step 2), the reverse transcriptases include the following: AMV reverse transcriptase, MMLV reverse transcriptase, PDB:1HMV reverse transcriptase.

6. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: In step 3), the method of removing the tissue includes the following methods: using proteinase K to digest the protein in the tissue and removing the tissue by washing with a solution; Trypsin was used to digest the proteins in the tissue and the tissue was removed by washing with solution; Collagenase was used to digest the proteins in the tissue and the tissue was removed by washing with solution.

7. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: In step 4), a primer probe targeting the cDNA after reverse transcription of the target mRNA is added for hybridization.

8. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 7, characterized in that: In step 4), the primer probes targeting different cDNAs contain different coding sequences.

9. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: The amplification method and fluorescent probe hybridization method include the following methods: the hybridization probe is circularized, RCA amplification is performed, and then a fluorescent probe is used for hybridization, and detection is performed by imaging; amplification is performed by the HCR method, and the probe has a fluorescent group and detection is performed by imaging; amplification is performed by the LAMP method, and then a fluorescent probe is used for hybridization and detection is performed by imaging.

10. The method for in situ sequencing of spatial transcriptome of tissue sections according to claim 1, characterized in that: In step 6), sequencing probes with different fluorophores are used for hybridization, and then the results of multiple rounds of hybridization and imaging are decoded to obtain the spatial distribution information of mRNA.

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