Space-time transcriptomics method

Abstract

The invention discloses a space-time transcriptome method which comprises the following steps: step 1, constructing a space transcriptome sequencing chip which is provided with a capture probe with space coordinates; step 2, carrying out metabolic marking by adopting a nucleotide analogue, freezing and embedding a slice after marking is completed, pasting the cut tissue slice on a sequencing chip, and carrying out fixed permeabilization so as to enable cells to release mRNA to be captured in situ by a capture probe on the space transcriptome sequencing chip; 3, carrying out treatment by using a nucleoside conversion reagent to cause base mutation in the corresponding cDNA, so as to distinguish the new RNA from the original RNA; and 4, analyzing the dynamic change of RNA under different external stimulation time.

Description

Technical Field

[0001] The present invention relates to a spatiotemporal transcriptomics method. Background Art

[0002] The spatial arrangement and dynamic changes of cells determine the function and fate of life tissues. Spatiotemporal transcriptomics can explore the spatial distribution, dynamic changes and interactions of gene expression in tissues, which is of great significance for in-depth understanding of complex biological processes and regulatory mechanisms in organisms. At present, spatial transcriptomics technology can couple transcriptome information with spatial coordinates in tissues, reconstruct cell distribution and molecular structure in the natural microenvironment of normal and diseased tissues, and study the functions and relationships of various cells with spatial locations. However, existing spatial transcriptome sequencing technologies can only obtain gene expression spectra at a certain moment, and cannot obtain dynamic RNA dynamics information. Existing metabolic labeling technologies still have some problems. Among them, the sci-fate technology has a low base T-to-C conversion rate during in situ conversion, while the SLAM-Drop-seq technology labeling process takes a long time and there will be interference from other substances.

[0003] Therefore, it is urgent to develop new spatiotemporal transcriptome sequencing technologies to analyze the dynamic changes of genes within tissues. Summary of the invention

[0004] In order to solve the problem of analyzing the dynamic changes of genes in tissues, the present invention provides a new spatiotemporal transcriptomics technology, which combines the existing spatial transcriptomics technology with metabolic labeling technology to analyze the spatiotemporal dynamic changes of genes in tissues by simultaneously analyzing new and old RNA in tissues.

[0005] The technical solution adopted by the present invention to solve its technical problem is:

[0006] A spatiotemporal transcriptomics method comprises the following steps:

[0007] Step 1, constructing a spatial transcriptome sequencing chip, wherein the spatial transcriptome sequencing chip is provided with capture probes with spatial coordinates;

[0008] Step 2: Metabolic labeling is performed using nucleotide analogs. After labeling, cryo-embedded sections are performed and the cut tissue sections are attached to the sequencing chip. The sections are fixed and permeabilized so that the mRNA released by the cells can be captured in situ by the capture probes on the spatial transcriptome sequencing chip.

[0009] Step 3: Treat with a nucleoside conversion reagent to cause base mutations in the corresponding cDNA, thereby distinguishing the new RNA from the original RNA;

[0010] Step 4: Analyze the dynamic changes of RNA under different external stimulation times.

[0011] Furthermore, step one includes the following steps:

[0012] (1) Modifying amino dendron molecules on epoxy chips;

[0013] (2) using microfluidics technology to modify two oligonucleotide sequences on amino resin molecules to form capture probes carrying spatial coordinates;

[0014] Furthermore, in step 2, the nucleotide analogs include but are not limited to 4-mercaptopurine (s4U), 5-ethynyluridine (5-EU), 5-bromouracil (5-BU), 5-fluorouracil (5-FU), 2-aminopurine (2-AP), 6-mercaptopurine (6-MP), etc.

[0015] Furthermore, step 2 includes the following steps:

[0016] (1) Use pepsin (or alkaline substances such as Triton and NaOH) to permeabilize tissues or cells to release RNA from tissues or cells;

[0017] (2) capturing RNA using the oligonucleotide probe modified in step 1;

[0018] Furthermore, in step three,

[0019] (1) The nucleoside conversion reagents include but are not limited to iodoacetamide (IAA), chloroacetamide (ClAA), iodoacetic acid and N-ethylmaleimide based on alkylation, hydrazine hydrate (N2H4·H2O) based on addition elimination, trifluoroethanol / sodium periodate (TFEA / NaIO4) based on oxidative nucleophilic aromatic substitution, etc.;

[0020] (2) Base mutations caused by nucleoside conversion reagents include but are not limited to T-to-C, T-to-G, T-to-A, C-to-T, A-to-G, A-to-C, A-to-T, etc.;

[0021] Furthermore, the entire process includes the following steps: modification of sequencing chips, metabolic labeling, tissue sectioning, tissue fixation, tissue permeabilization, nucleoside conversion, reverse transcription, second-strand synthesis, PCR amplification of cDNA, construction of sequencing libraries, and bioinformatics analysis of sequencing results.

[0022] Compared with the background technology, the technical solution of the present invention has the following advantages:

[0023] 1. Dynamic monitoring of RNA was achieved using metabolic labeling technology, and transcriptomic information was comprehensively analyzed from the spatiotemporal dimension.

[0024] Although spatial transcriptomic technology can obtain spatial information of RNA, it cannot obtain dynamic information of RNA changes over time, including RNA synthesis, degradation, shearing, etc. The present invention applies RNA metabolic labeling technology to spatial transcriptomic analysis, distinguishes newly synthesized RNA from the old RNA that existed in a period of time, obtains the spatial distribution of the newly synthesized RNA, and comprehensively analyzes the cell position, cell-to-cell interaction and gene regulation mechanism from the spatiotemporal dimension.

[0025] 2. It shortens the time required for traditional spatial transcriptomics experiments, simplifies the experimental process, and greatly reduces the difficulty of operation.

[0026] Directly modifying the capture probe on the glass slide, rather than first attaching the tissue and spatially encoding the mRNA in the tissue cells, reduces the difficulty of operation and avoids the RNA degradation problem caused by long-term exposure of tissue cells to the air.

[0027] 3. By changing the reaction conditions of IAA conversion, the mutation rate of base T-to-C was increased, and the time required for metabolic labeling and IAA conversion was shortened.

[0028] The high T-to-C mutation rate (increased to about 2%) makes the identification of nascent RNA more accurate. The time required for metabolic labeling and IAA conversion is shortened, reducing the risk of RNA degradation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] The present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0030] Figure 1 The figure is a flow chart of the preparation of the sequencing chip of the present invention.

[0031] Figure 2 The encoding principle of the sequencing chip and the composition of the capture probe sequence.

[0032] Figure 3 For 4 Principle of the nucleophilic substitution reaction of U with iodoacetamide.

[0033] Figure 4 This is a complete flow chart of Example 1.

[0034] Figure 5 is the mutation frequency of each mutation type under different solvent ratios.

[0035] Figure 6 is the mutation frequency of each mutation type under different IAA concentrations.

[0036] Figure 7 is the mutation frequency of each mutation type corresponding to different reaction times

[0037] Figure 8The optimized mutation frequency (T-to-C exceeds 2.0%)

[0038] Fig. 9 Different areas of the kidney.

[0039] Fig.10 The percentage of new RNA in different areas of the kidney. DETAILED DESCRIPTION

[0040] Example 1

[0041] Preparation of sequencing chips

[0042] 1. Modification of amino dendron molecules (PAMAM)

[0043] (1) Prepare 15 mL of 0.3% wt amino dendron molecule solution (the stock solution is PAMAM 6.0, 5% wt, Macklin, diluted with methanol), put a commercial epoxy-modified glass slide in a quartz cylinder, add the prepared amino dendron molecule solution, and place it on a shaker to react for about 14 hours;

[0044] (2) After the reaction, clean the glass slide surface with methanol and ultrapure water three times respectively, and blow dry with nitrogen gas.

[0045] 2. Chip orthogonal encoding

[0046] (1) Prepare 50 mM disuccinimidyl suberate (DSS, Thermo Scientific) solution;

[0047] (2) Replace Barcode X1-X 50 The stock solution (100 μM) was mixed with DSS solution and 1× PBS buffer (obtained from 10× dilution, invitrogen), and the final concentrations were: Barcode X 10 μM, DSS 2.5 mM;

[0048] (3) The microfluidic chip with transverse parallel channels is placed on the glass slide modified with amino resin molecules and fixed with a clamp. 3 μL of the 50 Barcode X solutions prepared above are added to the 50 injection ports in the corresponding order, and the channels are filled with the solution using a vacuum pump. The reaction is carried out in the dark for 1 hour.

[0049] (4) After the reaction, a vacuum pump was used to pass 0.1× SSC (obtained from 20× dilution, Invitrogen) to clean the channel, remove the chip, and block it with freshly prepared succinic anhydride (Solarbio, dissolved in N, N-dimethylformamide and triethylamine, final concentration 16% w / v) solution for 40 min. The glass slide was then washed with 0.1× SSC and ultrapure water;

[0050] (5) Barcoed Y1-Y 50 The stock solution was mixed with Ligation Linker 1:1 and annealed, and the annealed solution was mixed with T4 Ligation Buffer (Novagen), T4 DNA ligase (400 U / μL, Norvegant), and enzyme-free water to a final concentration of: Barcode Y 5 μM, T4 DNA ligase 40 U / μL;

[0051] (6) The microfluidic chip with longitudinal parallel channels is placed on a glass slide and fixed with a clamp. 3 μL of the 50 Barcode Y solutions prepared above are added to the 50 injection ports in the corresponding order, and the channels are filled with the solution using a vacuum pump. The reaction is carried out at 37°C for 30 min.

[0052] (7) After the reaction is completed, use a vacuum pump to pass 0.1×SSC to clean the channel, remove the chip, and clean the glass slide with ultrapure water.

[0053] Metabolic labeling and in situ RNA capture

[0054] 1.s 4 Metabolic labeling of U

[0055] (1) Prepare 80 mg / mL of s 4 U solution (4-mercaptouridine, Aldrich);

[0056] (2) Experimental mice (balb / c, female, about 8 weeks old) were given 1.0 mg (s 4 U / g (body weight) ratio was injected into the tail vein of the above-prepared 4 U solution, labeling for 1.5 h;

[0057] (3) After labeling, the kidneys were dissected and frozen and embedded;

[0058] (4) Use a Leica cryostat to slice tissue (slice thickness 10 μm) and attach the slices to the coding region of the sequencing chip.

[0059] 2. In situ capture, reverse transcription, and second-strand synthesis of mRNA

[0060] (1) The entire chip was immersed in methanol (pre-cooled at -20°C) for 30 min, stained with hematoxylin-eosin (H&E) staining, and imaged using a panoramic tissue slice scanner (Guangzhou Guangying Cell Technology Co., Ltd.);

[0061] (2) Prepare 0.1% pepsin solution (Pepsin, Sigma-Aldrich) with 0.1M HCl and incubate in a metal bath at 37°C for 15 min. Fix the chip with a clamp (specially designed, the tissue position forms a semi-enclosed space through a rubber ring, which is convenient for subsequent liquid addition operations), add the prepared pepsin solution (about 100 μL) into the rubber ring, seal it with dust-free tape, and perform PCR on a 37°C flat plate for 12 min. This step permeabilization changes the permeability of the cell membrane and nuclear membrane of the tissue cells, and the released mRNA is captured in situ by the capture probe containing spatial encoding information on the chip;

[0062] (3) Prepare 40 mM IAA solution (2-iodoacetamide, TCl) with DMSO (dimethyl sulfoxide) and 50 mM PB (phosphoric acid) solution, with the volume ratio of DMSO to 50 mM PB being 1:2. Remove the reagents from the previous step, wash with 0.1×SSC solution, add the prepared IAA solution, seal with dust-free tape, and perform PCR reaction at 37°C for 5 min;

[0063] (4) Prepare the reverse transcription mixture on ice: 5×RT buffer (final concentration 1×), Maxima H Minus Reverse Transcriptase (200U / μL, Thermo Scientific, final concentration 10U / μL), dNTPs (10mM, gold, final concentration 1mM), Recombinant RNase Inhibitor (40U / μL, Takara, final concentration 1U / μL), TemplateSwitch Oligo (50μM, final concentration 5μM), enzyme-free water, total volume 80μL. Remove the reaction reagents from the previous step, wash with 0.1×SSC solution, add the reverse transcription mixture, seal with dust-free tape, and react on a plate at 42°C for 2h;

[0064] (5) Prepare the second-strand synthesis mixture on ice: 10×Isothermal buffer (NEB, final concentration 1×), MgSO4 (100mM, NEB, final concentration 6mM), Bst2.0 polymerase (8000U / mL, NEB, final concentration 320U / mL), dNTPs (10mM, full gold, final concentration 1.4mM), Primers (100mM, final concentration 10mM), enzyme-free water, total volume 75μL. Remove the reaction reagents from the previous step, wash with 0.1×SSC solution, add 0.08M NaOH and let stand at room temperature for 5min, remove NaOH, wash with EBElution Buffer (Qiagen), add the second-strand synthesis mixture, seal with dust-free tape, set the plate PCR program: 37℃15min, 45℃15min, 55℃15min, 65℃15min, store at 4℃, and start the program;

[0065] (6) After the reaction, remove the reagents, add 36 μL 0.08 M NaOH and let stand at room temperature for 10 min. Recover the mixture into a tube, add 5 μL Tris-HCl (pH 7.0, Invitrogen) to neutralize, and add 40 μL 2× KAPA HiFi Mix (Roche, final concentration 1×) and two corresponding adapter sequences for PCR amplification.

[0066] 3. Construction of sequencing library and NGS sequencing

[0067] The sequencing library was constructed using the Watchmaker DNA Library Prep Kit with Fragmentation product and sequenced on a sequencing machine.

[0068] Experimental Results

[0069] 1. Optimization of IAA conversion conditions

[0070] The ratio of the two solvents DMSO and PB in the reaction system, the concentration of IAA and the reaction time were optimized and compared. After optimization (DMSO:PB = 1:2, IAA concentration of 40mM, reaction time of 5min), the base T-to-C mutation rate increased to 2.0%. Figure 5 .

[0071] The experiment was conducted with the ratio of DMSO to PB set at 1:8, 1:4, and 1:2. The results showed that when DMSO:PB=1:2, the T-to-C mutation rate was much higher than that of other conditions, reaching 2.4%. Figure 6 .

[0072] Subsequently, the concentrations of IAA were set to 10mM, 40mM, and 80mM for comparative experiments. The results showed that the T-to-C mutation frequency produced by 40mM and 80mM IAA treatments was almost unchanged (close to 2.4%), indicating that 40mM IAA had saturated the reaction conditions. Subsequent experiments were all carried out under the condition of 40mM IAA. Figure 7 .

[0073] Next, we explored the effect of reaction time on IAA conversion results. As can be seen from the figure, when the reaction time is 5 minutes, the T-to-C mutation rate exceeds 1.2% (since further increasing the reaction time is likely to cause RNA degradation, the IAA conversion time of subsequent experiments is set to 5 minutes). Figure 8 .

[0074] The final optimized conditions were used for formal experiments, and the mutation frequency of base T-to-C was about 2.3%.

[0075] 2. Spatiotemporal analysis of nascent RNA

[0076] According to the difference of tissue cell markers, the renal tissue is divided into five different areas from the outside to the inside: cortex, outer medulla (including outer band and inner band), inner medulla and renal pelvis (see Fig. 9 ). The newly generated RNA in different regions was analyzed separately to obtain the corresponding proportion of newly generated RNA (since the newly generated RNA was mainly concentrated in other regions except the renal pelvis, the renal pelvis region was not calculated during the statistics). It was found that the proportion of newly generated RNA in each region was between 20% and 30%, with the proportion of newly generated RNA in the inner band and inner medulla being relatively high, close to 30%, and the proportion of newly generated RNA in the cortex being relatively low, at around 21%. Fig.10 .

[0077] 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 spatiotemporal transcriptomics method comprising the following steps: Step 1, constructing a spatial transcriptome sequencing chip, wherein the spatial transcriptome sequencing chip is provided with capture probes with spatial coordinates; Step 2: Metabolic labeling is performed using nucleotide analogs. After labeling, cryo-embedded sections are performed and the cut tissue sections are attached to the sequencing chip. The sections are fixed and permeabilized so that the mRNA released by the cells can be captured in situ by the capture probes on the spatial transcriptome sequencing chip. Step 3: Treat with a nucleoside conversion reagent to cause base mutations in the corresponding cDNA, thereby distinguishing the new RNA from the original RNA; Step 4: Analyze the dynamic changes of RNA under different external stimulation times.

2. A spatiotemporal transcriptome method according to claim 1, characterized in that: Step 1 includes the following steps: (1) Modifying amino dendron molecules on epoxy chips; (2) Microfluidics technology is used to modify two oligonucleotide sequences on amino resin molecules to form capture probes carrying spatial coordinates.

3. A spatiotemporal transcriptomics method according to claim 1, characterized in that: In step 2, the nucleotide analogue includes at least one of 4-mercaptopurine, 5-ethynyluridine, 5-bromouracil, 5-fluorouracil, 2-aminopurine and 6-mercaptopurine.

4. A spatiotemporal transcriptomics method according to claim 1, characterized in that: Step 2 includes the following steps: (1) Permeabilizing tissues or cells using pepsin or alkaline substances to release RNA from tissues or cells; (2) Capture RNA using the oligonucleotide probe modified in step 1.

5. A spatiotemporal transcriptome method according to claim 1, characterized in that: In step three, the base mutation caused by the nucleoside conversion reagent includes at least one of T-to-C, T-to-G, T-to-A, C-to-T, A-to-G, A-to-C, and A-to-T.

6. A spatiotemporal transcriptomics method according to claim 1, characterized in that: In step 3, the nucleoside conversion reagent includes at least one of iodoacetamide IAA, chloroacetamide, iodoacetic acid and N-ethylmaleimide based on alkylation, hydrazine hydrate based on addition elimination, and trifluoroethanol / sodium periodate based on oxidative nucleophilic aromatic substitution.

7. A spatiotemporal transcriptome method according to claim 6, characterized in that: In the preparation of iodoacetamide IAA, the ratio of DMSO to PB is 1:(3.9-4.1).

8. A spatiotemporal transcriptomics method according to claim 6, characterized in that: The IAA concentration is 40mM-80mM.

9. A spatiotemporal transcriptomics method according to claim 6, characterized in that: The IAA reaction time is 4.5-5.5min.

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