WO2024138523A1

WO2024138523A1 - Spatiotemporal transcriptomic sequencing method

Info

Publication number: WO2024138523A1
Application number: PCT/CN2022/143374
Authority: WO
Inventors: 赵宇; 刘伟庆; 李兆勋; 向浩鑫; 廖莎; 江霞; 袁巧梅; 陈冬冬; 周慧; 陈奥; 章文蔚
Original assignee: 深圳华大生命科学研究院
Filing date: 2022-12-29
Publication date: 2024-07-04

Abstract

Provided in the present disclosure is a spatiotemporal transcriptomic sequencing method, comprising: determining the cDNA abundance of a sample to be sequenced; and performing a spatiotemporal transcriptomic sequencing step on said sample on the basis of the cDNA abundance, wherein determining the cDNA abundance of said sample comprising a pre-spatiotemporal transcriptomic sequencing step. According to the specific embodiments of the present disclosure, the cDNA abundance obtained by means of the method can accurately reflect the gene capture capability of the spatiotemporal transcriptomic technology on tissue slices. Compared with the prior art, the method omits steps of cDNA recovery and cDNA shearing, thereby greatly saving the operation time.

Description

Spatiotemporal transcriptome sequencing methods

Technical Field

The present disclosure relates to the field of biotechnology, and in particular, to a spatiotemporal transcriptome sequencing method.

Background technique

In multicellular organisms, gene expression in individual cells strictly follows a specific temporal and spatial order, that is, gene expression has temporal and spatial specificity. Temporal specificity can be analyzed by taking samples at different time points and using single-cell transcriptome sequencing technology to analyze cell types and gene expression patterns in the temporal dimension. Spatial specificity information is relatively difficult to obtain. The high-throughput spatial transcriptome technology developed in recent years can simultaneously obtain gene expression characteristics and spatial distribution data in tissue situ, further advancing the study of true gene expression of cells in situ in tissues.

At present, spatiotemporal transcriptomics technology has certain requirements for the RNA quality of samples. For frozen OCT samples, the RNA quality needs to be tested with Agilent RNA 6000 Pico Kit, and samples with RIN ≥ 7 can be used for subsequent experiments; for FFPE samples, the RNA quality needs to be tested with Agilent RNA 6000 Pico Kit, and samples with DV200 ≥ 50% can be used for subsequent experiments.

Therefore, the optimization of spatiotemporal transcriptomics technology needs to be studied.

Summary of the invention

The present disclosure aims to solve at least one of the technical problems existing in the prior art to at least some extent.

During the experiment, the inventors found that the final number of captured genes and RNA quality detection using Agilent RNA 6000 Pico Kit were not completely positively correlated (Table 1). For example, some FFPE samples with DV200 ≥ 50% (such as mouse tongue and mouse thymus) still had lower capture than other samples with DV200 ≥ 50%.

Table 1

Therefore, in view of the problem that the above-mentioned RNA quality inspection cannot accurately reflect the capture ability of the spatiotemporal transcriptome for samples, the inventors designed a new quality detection method for spatiotemporal transcriptome samples. This method extracts RNA from tissue sections and then reverse transcribes it, then enriches it by PCR to obtain a cDNA library; then, through second-generation sequencing, the abundance of the cDNA in the library is analyzed to determine the actual capture ability of the spatiotemporal transcriptome technology for the sample section, and then analyze whether the sample is suitable for the spatiotemporal transcriptome technology.

The steps of the new spatiotemporal transcriptome quality inspection method are as follows: (1) First, the tissue section to be tested is attached to a glass slide or a bare chip (a chip without a capture probe), fixed and permeabilized, and then a random probe 6N with a fixed sequence at the 5' end is added to hybridize with the tissue section. The six random bases at the 3' end of the random probe 6N will capture RNA (as shown in ① in Figure 1); then, a reverse transcription (RT) reaction solution is added, and the random probe 6N polymerizes cDNA using RNA as a template (as shown in ② in Figure 1); while RT is being performed, TSO is added through the terminal transferase activity of the RT enzyme, which is adapter 2, and the 5' fixed sequence on the random probe 6N is adapter 1. Through these two adapters, PCR amplification is performed to obtain a cDNA library (as shown in ③ in Figure 1). The obtained library is then sequenced using the second-generation sequencing technology. (2) The obtained sequencing results are subjected to bioinformatics analysis, and the specific process is shown in Figure 2. The obtained .fastq file (a file format for storing biological sequences (usually nucleotide sequences) and their quality values) is aligned to the reference genome of the tissue using STAR software, and a .bam alignment file is obtained. Then, the .bam alignment results are annotated using Bam2Gem, a self-developed annotation tool of BGI, and the annotated files are filtered using Samtools. Finally, the cDNA abundance curve is drawn with the number of filtered annotated reads as the horizontal axis and the number of gene types as the vertical axis.

Therefore, in one aspect of the present disclosure, the present disclosure proposes a spatiotemporal transcriptome sequencing method. According to an embodiment of the present disclosure, the method includes determining the cDNA abundance of the sample to be sequenced; and based on the cDNA abundance, performing a spatiotemporal transcriptome sequencing step on the sample to be sequenced; wherein, the determination of the cDNA abundance of the sample to be sequenced includes a pre-spatiotemporal transcription sequencing step. According to a specific embodiment of the present disclosure, the cDNA abundance obtained by this method can accurately reflect the ability of spatiotemporal transcriptome technology to capture tissue section genes. Compared with the prior art, the steps of cDNA recovery and cDNA shearing are omitted, which greatly saves operation time.

According to an embodiment of the present disclosure, the above-mentioned spatiotemporal transcriptome sequencing method may further include at least one of the following additional technical features:

According to an embodiment of the present disclosure, the sample to be tested is a tissue sample.

According to the embodiment of the present disclosure, the pre-spatial transcription sequencing step includes: PCR amplification of the sample to be tested, the sample to be tested has been hybridized with a probe and reverse transcribed in advance; and sequencing of the PCR amplification product. By adopting the pre-spatial transcription sequencing method, compared with the prior art, the steps of cDNA recovery and cDNA shearing are omitted, making the workflow simpler and greatly saving time costs.

According to an embodiment of the present disclosure, sequencing the PCR amplification product includes: performing a cyclization treatment on the PCR amplification product to obtain a DNB library; and sequencing the DNB library. By adopting the pre-temporal and spatial transcription sequencing method, compared with the prior art, the steps of cDNA recovery and cDNA shearing are omitted, making the workflow simple and greatly saving time costs.

According to an embodiment of the present disclosure, the method further includes: performing comparison processing on the sequencing processing results to obtain the cDNA abundance of the sample to be sequenced. By adopting the pre-temporal and spatial transcription sequencing method, compared with the prior art, the steps of cDNA recovery and cDNA shearing are omitted, making the workflow simpler and greatly saving time costs.

According to the embodiments of the present disclosure, the probe is a random probe 6N, a poly-T with a fixed sequence at 5', or a targeted primer with a fixed sequence at 5'. The inventors found that these probes can bind to RNA in tissue sections through complementary base pairing to form hybrid molecules.

According to an embodiment of the present disclosure, the random probe 6N includes the nucleotide sequence shown in SEQ ID NO: 1. Thus, the tissue section can be bound by the primer on the probe, so that the probe can capture adjacent mRNA from the tissue.

5’-CCTCCGACTGTGTGACTTAGACTTGCACTTGATGTGCTNNNNNN-3’ (SEQ ID NO: 1)

According to an embodiment of the present disclosure, the sequencing process is performed on an MGISEQ-2000RS platform, a qPCR or a multiplex PCR platform.

According to an embodiment of the present disclosure, the comparison process includes: first comparing the sequencing result with the reference genome; annotating the first comparison result to obtain a first annotation result; screening the first annotation result to obtain a second annotation result; based on the number of annotated genes in the second annotation result and the predetermined number of annotated genes, obtaining the cDNA abundance of the sample to be sequenced. The cDNA abundance obtained by the comparison process can accurately judge whether the tissue sample is suitable for spatiotemporal transcriptome technology. Compared with the existing RNA quality inspection, the cDNA abundance can more accurately reflect the problem of the spatiotemporal transcriptome's ability to capture samples.

According to an embodiment of the present disclosure, the first comparison is performed by STAR software.

According to an embodiment of the present disclosure, the annotation is performed by Bam2Gem.

According to an embodiment of the present disclosure, the screening process is performed by Samtools.

According to the embodiment of the present disclosure, the PCR amplification is performed in the presence of adapter primer 1 and adapter primer 2. By introducing adapter primers 1 and 2, it is possible to complement the two wings of the target DNA segment to be amplified, so that the template DNA sequence can be effectively amplified.

According to an embodiment of the present disclosure, the linker primer 1 includes the nucleotide sequence shown in SEQ ID NO: 2, wherein the 5' end of the linker primer 1 is phosphorylated. At this time, the nucleotide sequence included in the linker primer 1 can be recorded as 5'-pho-CTGCTGACGTACTGAGAGGC-3'.

5’-CTGCTGACGTACTGAGAGGC-3’ (SEQ ID NO: 2)

According to an embodiment of the present disclosure, the linker primer 2 includes the nucleotide sequence shown in SEQ ID NO: 3, wherein the 5' end of the linker primer 2 is not phosphorylated.

5’-GAGACGTTCTCGACTCAGCAGA-3’ (SEQ ID NO: 3)

According to the embodiment of the present disclosure, the procedure of the PCR amplification is: ① 90-98°C for 2-7 minutes, ② 95-99°C for 10-30 seconds, ③ 55-60°C for 10-30 seconds, ④ 70-75°C for 1-5 minutes, ②-④ for 15 cycles, 70-75°C for 2-7 minutes. By adopting this procedure, the starting template of the PCR reaction can be completely denatured, avoiding the inability to effectively utilize the template in the first amplification cycle, resulting in a low amplification yield.

According to the embodiment of the present disclosure, the PCR amplification procedure is: 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, 72°C for 5 minutes. By adopting this procedure, the starting template of the PCR reaction can be completely denatured, avoiding the inability to effectively utilize the template in the first amplification cycle, resulting in a low amplification yield.

According to an embodiment of the present disclosure, the tissue sample is sliced in advance.

According to an embodiment of the present disclosure, the slicing product is further subjected to chip attaching, cross-linking removal, solidification and permeabilization treatment, so as to release mRNA from the cells.

According to an embodiment of the present disclosure, the spatiotemporal transcriptome sequencing step includes: performing PCR amplification on the sample to be tested, wherein the sample to be tested has been hybridized with a probe and reverse transcribed in advance; and sequencing the PCR amplification product. Compared with the prior art, the spatiotemporal transcriptome sequencing method omits the steps of cDNA recovery and cDNA shearing, greatly saving operation time.

According to an embodiment of the present disclosure, the step of sequencing the PCR amplification product includes: performing a cyclization treatment on the PCR amplification product to obtain a DNB library; and sequencing the DNB library. Compared with the prior art, the spatiotemporal transcriptome sequencing method omits the steps of cDNA recovery and cDNA shearing, greatly saving operation time.

Additional aspects and advantages of the present disclosure will be given in part in the following description and in part will be obvious from the following description or learned through practice of the present disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and/or additional aspects and advantages of the present disclosure will become apparent and easily understood from the description of the embodiments in conjunction with the following drawings, in which:

FIG1 is a schematic diagram of a novel RNA quality inspection method according to Example 2 of the present disclosure - a schematic diagram of obtaining cDNA abundance (biochemical part);

FIG2 is a schematic diagram of a novel RNA quality inspection method according to Example 2 of the present disclosure - a schematic diagram of obtaining cDNA abundance (bioinformatics part), (wherein, *fastq: a file format for storing biological sequences (usually nucleotide sequences) and their quality values; Bam2Gem: an annotation tool developed by BGI, used to annotate alignment results to the genome; Samtools: a tool for reading .bam files);

Figure 3 is a comparison chart of the spatiotemporal transcriptome capture results of FFPE mouse kidney, FFPE mouse brain and FFPE mouse lung according to Example 1 of the present disclosure (A) and the spatiotemporal transcriptome capture results of FFPE mouse kidney, FFPE mouse brain and FFPE mouse lung detected by the method of Example 2 (B) (the horizontal axis of Figure B represents the sequencing depth, i.e., the number of sequencing reads; the vertical axis represents the number of gene types obtained using the cDNA abundance analysis method).

Detailed ways

The embodiments of the present disclosure are described in detail below. The embodiments described below are exemplary and are only used to explain the present disclosure, and should not be construed as limiting the present disclosure. If no specific techniques or conditions are specified in the embodiments, the techniques or conditions described in the literature in this field or the product instructions are used. If the manufacturer of the reagents or instruments used is not specified, they are all conventional products that can be obtained commercially.

Example 1

1. Sample sectioning

Paraffin sample sectioning: Use a Leica paraffin slicer (HistoCore BIOCUT), set the paraffin section thickness to 5 μm, and slice after appropriately trimming the paraffin-embedded blocks.

2. RNA quality control

Paraffin samples: Cut 2-4 5μm slices and place them in RNase-free 1.5ml centrifuge tubes. Use the RNeasy FFPE kit to extract RNA and use the Agilent RNA 6000 Pico Kit to detect RNA quality. Samples with DV200 ≥ 50% can be used for subsequent experiments.

3. Chip processing and tissue patching

Carefully pick up the capture chip with tweezers, place it in a new 24-well plate, and record the capture chip number. The capture chip contains spatial barcode information and fixed oligonucleotide sequence: 5'-NNNNNNNNNNNNNNNNNNNNNNNNNTTGTCTTCCTAAGACCGCTTGG-3' (SEQ ID NO: 4). Wash the chip twice with 400 μL 0.1×SSC, clean it with nuclease-free pure water (NF-H ₂ O), and dry the chip at 37°C. Cut the paraffin sample into 5-8 μm, spread it in a 42°C water bath, pick it up with the capture chip and bake it at 60°C, then place the chip with the tissue in the dewaxing reagent for 30 minutes, repeat once; remove it and dry it on absorbent paper, place it in 100% anhydrous ethanol for 5 minutes, repeat once; remove it and place it in 96% ethanol for 5 minutes, repeat once.

4. Tissue Section Decrosslinking, Fixation and Permeabilization

After the chip is dried, put it in a 24-well cell plate, add cross-linking decontamination buffer (380μl TE buffer at pH 9.0 + 20μl RNase Inhibitor (RI) and mix well), and react in a 70℃ incubator for 1 hour. After the reaction is completed, take out the chip, wait for the chip to return to room temperature, and fix it in a -20℃ precooled methanol solution for 20 minutes. After taking it out, dry the methanol, add the permeabilization reagent (same as above), and react in a 37℃ incubator for 20 minutes.

5. Hybridization of random probe 6N with tissue and reverse transcription (RT)

Hybridization of random probe 6N with tissue: 5μM random probe 6N (5’-CCTCCGACTGTGTGACTTAGACTTGCACTTGATGTGCTNNNNNN-3’ (SEQ ID NO: 1)) was dissolved in 5×SSC solution, 5% volume of RI was added, and mixed. The permeabilized tissue sections were washed once with 5×SSC+RI, and then the mixed random probe 6N solution was added and hybridized at room temperature for 15 minutes. In this process, random probe 6N will capture RNA in the tissue. Then wash once with 5×SSC+RI.

Reverse transcription (RT) reaction: Add RT reaction solution to the chip surface and react at 42°C for 3 hours. Remove and wash once with 0.1×SSC+RI.

6.Splint hybridization and T4 ligation

Splint hybridization: dilute 5μM splint (5’-CAAGTGCAAGTCTAAGTCACACAGTCGGAGGCCAAGCGGTCTTAG-3’ (SEQ ID NO: 5)) into 5×SSC solution, add 5% volume of RI, mix well, add to the slices washed in the previous step, and hybridize at room temperature for 15 minutes.

T4 ligation: Add T4 ligation reaction solution and react at 16°C overnight.

7. Tissue Removal

After the reaction, the chip was taken out, the surface liquid was sucked dry, and it was washed once with NF-H ₂ O. Tissue removal solution was added, and the chip was placed in a 55°C incubator for reaction for 20 minutes. After taking it out, the chip surface was sucked and then sucked twice with NF-H ₂ O to remove the tissue.

8.cDNA recovery, amplification and purification

cDNA recovery: add 400μl/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 circle to prevent volatilization, react in a 55℃ incubator for 3h to release cDNA; recover the liquid in the reaction well into a new 1.5ml centrifuge tube, add 350μl/well NF-H ₂ O to clean the chip, and recover the cleaning solution into the same centrifuge tube. Add VAHTSTM DNA Clean Beads (VAZYME) to the recovery solution in a ratio of 0.8:1 in a 1.5ml centrifuge tube, shake and mix, and incubate at room temperature for 10 minutes. After instant centrifugation, place the EP tube on a magnetic stand and let it stand for 3 minutes. After the liquid is clarified, remove the supernatant, add 1ml of freshly prepared 80% ethanol, shake and mix, centrifuge instantly, and let it stand for 30 seconds; discard the supernatant, and repeat the washing with 80% ethanol. Discard the supernatant and dry at room temperature until the surface of the magnetic beads is non-reflective and cracked; add 42μl NF-H2O to dissolve, shake and mix, let stand at room temperature for 5 minutes, centrifuge briefly, let stand on the magnetic rack for 3-5 minutes, and recover the supernatant into a new PCR tube after the liquid is clarified.

cDNA amplification: Add 58μl PCR mixture (including cDNA HIFI Master Mix 50μl, cDNA Primers 8μl) to the above PCR tube, a total of 100μl, and perform PCR reaction. The PCR reaction steps are: ①95℃ 5 minutes, ②98℃ 20 seconds, ③58℃ 20 seconds, ④72℃ 3 minutes, ②～④ react for 15 cycles, then 72℃ 5 minutes, take out and place on ice. Use Qubit dsDNA Mix to detect the concentration of cDNA after amplification: InvitrogenTM Qubit dsDNA HS Buffer 198μl, Qubit dsDNA HS Reagent 200×1μl, cDNA product 1μl.

cDNA purification: Transfer the PCR product from the previous step to a new 1.5ml centrifuge tube, mix it with VAHTSTM DNA Clean Beads (VAZYME) equilibrated at room temperature in a volume ratio of 1:1, shake and mix, and incubate at room temperature for 10 minutes. After instant centrifugation, place the EP tube on a magnetic stand for 3 minutes, and remove the supernatant after the liquid is clear. Add 1ml of freshly prepared 80% ethanol, let it stand for 30 seconds, discard the supernatant, repeat the washing with 80% ethanol once, discard the supernatant, and dry it at room temperature until the surface of the magnetic beads is not reflective or cracked. Add 40μl NF-H ₂ O to dissolve it back, shake and mix it, let it stand at room temperature for 5 minutes, centrifuge it instantaneously, let it stand on a magnetic stand for 3-5 minutes, and after the liquid is clear, recover the supernatant into a new 1.5ml centrifuge tube. Take 1μl of cDNA sample and use Qubit dsDNA HS Kit to detect the concentration, and use Agilent 2100 High Sensitivity DNA kit to detect the distribution of cDNA fragments.

9.cDNA shearing, amplification and purification

cDNA shearing: Prepare shearing reagents, 4μl 5×TAG buffer, 1μl shearing enzyme, 20ng cDNA purification product, make up to 20μl with NF-H ₂ O, react at 55℃ for 10 minutes, and then immediately put on ice. Add 5μl Stop buffer to the shearing product and let it stand at room temperature for 5 minutes.

Interrupt product amplification: Add 50μl Library HIFI Master Mix and 25μl Library PCR Primer Mix to the above reaction solution, mix well and then perform PCR reaction. The PCR reaction steps are: ①95℃ for 5 minutes, ②98℃ for 20 seconds, ③58℃ for 20 seconds, ④72℃ for 30 seconds, ②~④ for 13 cycles, then 72℃ for 5 minutes, take out and place on ice.

Purification of amplification products: 100 μl of the above amplification products were added with 60 μl of VAHTSTM DNA Clean Beads (VAZYME) equilibrated at room temperature, vortexed and mixed, incubated at room temperature for 5 minutes, centrifuged briefly and placed on a magnetic stand for 3 minutes, and the supernatant was transferred to a new PCR tube after the liquid was clarified. 20 μl of Vazyme Beads were added to the supernatant and mixed with the supernatant, vortexed and mixed, incubated at room temperature for 5 minutes, centrifuged briefly and placed on a magnetic stand, and placed on a magnetic stand for 3-5 minutes until the liquid was clarified, and the supernatant was carefully aspirated and discarded with a pipette. 200 μl of freshly prepared 80% ethanol was added, and the supernatant was carefully aspirated and discarded, and the 80% ethanol was used to wash once again, and the magnetic beads were dried at room temperature until the surface of the magnetic beads was not reflective or cracked. 20 μl of NF-H ₂ O was added to dissolve, vortexed and mixed, and placed on a magnetic stand for 5 minutes at room temperature, centrifuged briefly and placed on a magnetic stand for 3 minutes, and the supernatant was transferred to a new centrifuge tube after the liquid was clarified. Take 1 μl of the purified product and use Qubit dsDNA HS Kit to detect the concentration, and use Agilent 2100 High Sensitivity DNA kit to detect the fragment distribution.

10. cDNA library circularization, sequencing, and data analysis

Take 40 ng of the above purified product into a new PCR tube, add NF-H ₂ O to make it up to 20 μl, add 20 μl 2×Make DNB buffer, and react in a PCR instrument: 95°C for 3 minutes, 40°C for 3 minutes. After taking out the above reaction solution, place it on ice, add 39 μl RCA buffer, 1 μl 10mM ATP, 4 μl One Step Enzyme, react in a PCR instrument at 30°C for 30 minutes, take it out and add 20 μl DNB stop buffer, which is the circularized DNB library. The library was sequenced on the MGISEQ-2000RS sequencer. The offline data was automatically analyzed on the https://uat.stomics.tech/sap/ website, and the heat map results were finally obtained (as shown in A in Figure 3).

Example 2

1. Sample sectioning

Use a Leica paraffin slicer (HistoCore BIOCUT), set the paraffin section thickness to 5 μm, and trim the paraffin-embedded blocks appropriately before slicing.

2. Chip processing and tissue patching

Use bare chips (or glass slides) treated with NF-H ₂ O. Cut the paraffin sample into 5-8μm pieces, spread the slices in a 42℃ water bath, pick up the slices with bare chips and bake them at 60℃, then place the slices with tissues in the dewaxing reagent for 30 minutes, repeat once; take them out and dry them on absorbent paper, place them in 100% anhydrous ethanol for 5 minutes, repeat once; take them out and place them in 96% ethanol for 5 minutes, repeat once.

3. Tissue Section Decrosslinking, Fixation and Permeabilization

4. Hybridization of random probe 6N with tissue and reverse transcription (RT)

5μM random probe 6N (5’-CCTCCGACTGTGTGACTTAGACTTGCACTTGATGTGCTNNNNNN-3’ (SEQ ID NO: 1)) was dissolved in 5×SSC solution, and 5% volume of RI was added and mixed. The permeabilized tissue sections were washed once with 5×SSC+RI, and then the mixed random probe 6N solution was added and hybridized at room temperature for 15 minutes. Then RT reaction solution was added to the chip surface and reacted at 42°C for 3 hours. After taking out, it was washed once with 0.1×SSC+RI.

5. ChIP-PCR

Split the chip into thin strips that can be put into a PCR tube, add PCR premix (containing primers for adapter 1 and adapter 2, adapter 1: 5’-pho-CTGCTGACGTACTGAGAGGC-3’ (SEQ ID NO: 2); adapter 2: 5’-GAGACGTTCTCGACTCAGCAGA-3’ (SEQ ID NO: 3)), and react in a PCR instrument. The procedure is: ① 95℃ for 5 minutes, ② 98℃ for 20 seconds, ③ 58℃ for 20 seconds, ④ 72℃ for 3 minutes, ② to ④ react for 15 cycles, then 72℃ for 5 minutes, take out and place on ice.

6. cDNA library circularization and sequencing

Take 40ng of the above purified product into a new PCR tube, add NF-H ₂ O to make up to 20μl, add 20μl 2×Make DNB buffer, and react in a PCR instrument: 95℃ for 3 minutes, 40℃ for 3 minutes. After taking out the above reaction solution, place it on ice, add 39μl RCA buffer, 1μl 10mM ATP, 4μl One Step Enzyme, react in a PCR instrument at 30℃ for 30 minutes, take it out and add 20μl DNB stop buffer, which is the circularized DNB library. The library is sequenced on the MGISEQ-2000RS sequencer. (NF-H ₂ O, 2×Make DNB buffer, RCA buffer, ATP, One Step Enzyme and DNB stop buffer are all from the high-throughput sequencing primer kit (spatiotemporal omics) with catalog number 940-000037-00, MGI)

7. Data Analysis

The off-machine results were analyzed using the process shown in Figure 2 to obtain the final cDNA abundance curve.

8. Based on the obtained cDNA abundance, perform spatiotemporal transcriptome analysis of the sample to be tested - repeat steps 1 to 6, and sequence the library using the MGISEQ-2000RS sequencer. The data is automatically analyzed on the https://uat.stomics.tech/sap/ website. The analysis results are shown in Figure 3.

Example 3

The method in Example 1 was used to capture gene information in tissue sections of rat kidney, rat brain and rat lung; the method in Example 2 was used to obtain cDNA abundance and gene quantity in tissue sections of rat kidney, rat brain and rat lung. Among them, when the rat kidney, rat brain and rat lung were detected by Example 2, the number of genes detected was 20465, 19856 and 16816 respectively when the sequencing depth was 0.2×10 ⁸ , i.e. 20M reads. The comparison results of Example 1 and Example 2 are shown in Figure 3, where Figure 3A is the gene capture result obtained by the method of Example 1 for FFPE rat kidney, FFPE rat brain and FFPE rat lung; Figure 3B is the number of gene capture detected by the method of Example 2 for rat kidney, rat brain and rat lung. By comparing the results of the two methods, it was found that when the method of Example 2 was used to perform spatial transcriptome sequencing analysis on the test sample, that is, the RNA quality of the test sample was quality checked by determining the cDNA abundance and then the spatial transcription analysis was performed on the test sample. The cDNA abundance can accurately reflect the ability of spatiotemporal transcriptome technology to capture genes in tissue sections, and the final number of captured genes is positively correlated with the RNA quality detection.

In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "example", "specific example", or "some examples" etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present disclosure. In this specification, the schematic representations of the above terms do not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner. In addition, those skilled in the art may combine and combine different embodiments or examples described in this specification and the features of different embodiments or examples, unless they are contradictory.

Although the embodiments of the present disclosure have been shown and described above, it is to be understood that the above embodiments are illustrative and are not to be construed as limitations of the present disclosure. A person skilled in the art may change, modify, replace and vary the above embodiments within the scope of the present disclosure.

Claims

A spatiotemporal transcriptome sequencing method, characterized by comprising:

Determine the abundance of cDNA in the sample to be sequenced; and

Based on the cDNA abundance, performing a spatiotemporal transcriptome sequencing step on the sample to be tested;

Wherein, the determination of the cDNA abundance of the sample to be sequenced includes a pre-spatiotemporal transcription sequencing step.
The method according to claim 1, characterized in that the sample to be tested is a tissue sample.
The method according to claim 1, characterized in that the pre-spatial transcription sequencing step comprises:

Amplifying the sample to be tested by PCR, wherein the sample to be tested has been subjected to hybridization with a probe and reverse transcription treatment in advance; and

The PCR amplification product is sequenced.
The method according to claim 3, characterized in that sequencing the PCR amplification product comprises:

Circularizing the PCR amplification product to obtain a DNB library; and

The DNB library is sequenced.
The method according to claim 3 or 4, further comprising:

The sequencing results are compared to obtain the cDNA abundance of the sample to be sequenced.
The method according to claim 3, characterized in that the probe is a random probe 6N, a poly-T with a fixed sequence at 5', or a targeted primer with a fixed sequence at 5';

Optionally, the random probe 6N includes the nucleotide sequence shown in SEQ ID NO: 1.
The method according to claim 3 or 4, characterized in that the sequencing process is performed on an MGISEQ-2000RS platform, a qPCR or a multiplex PCR platform;

Optionally, the comparison process includes:

Performing a first comparison of the sequencing results with a reference genome;

Annotating the first comparison result to obtain a first annotation result;

Screening the first annotation result to obtain a second annotation result;

Based on the number of annotated genes in the second annotation result and the predetermined number of annotated genes, obtaining the cDNA abundance of the sample to be sequenced;

Optionally, the first alignment is performed by STAR software;

Optionally, the annotation is performed by Bam2Gem;

Optionally, the screening process is performed by Samtools.
The method according to claim 3 is characterized in that the PCR amplification is carried out in the presence of linker primer 1 and linker primer 2.
The method according to claim 8, characterized in that the adapter primer 1 comprises the nucleotide sequence shown in SEQ ID NO: 2, wherein the 5' end of the adapter primer 1 is phosphorylated;

Optionally, the adapter primer 2 comprises the nucleotide sequence shown in SEQ ID NO: 3, wherein the 5' end of the adapter primer 2 is not phosphorylated;

Optionally, the PCR amplification program is: ① 90-98°C for 2-7 minutes, ② 95-99°C for 10-30 seconds, ③ 55-60°C for 10-30 seconds, ④ 70-75°C for 1-5 minutes, ②-④ for 15 cycles, 70-75°C for 2-7 minutes;

Preferably, the PCR amplification program is: 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 react for 15 cycles, and 72°C for 5 minutes.
The method according to claim 2, characterized in that the tissue sample is previously sliced;

Optionally, the process further includes subjecting the sliced product to chip attaching, cross-linking removal, curing and permeabilization.
The method according to claim 1, characterized in that the spatiotemporal transcriptome sequencing step comprises:

Amplifying the sample to be tested by PCR, wherein the sample to be tested has been hybridized with a probe and reverse transcribed in advance;

The PCR amplification product was sequenced.
The method according to claim 11, characterized in that sequencing the PCR amplification product comprises:

The PCR amplification product is subjected to circularization treatment to obtain a DNB library;

And sequencing the DNB library.