High-throughput single cell space transcriptome method based on multi-round imaging coding

Abstract

The invention relates to a single cell space transcriptome method based on multi-round imaging coding. The single cell space transcriptome method comprises the following steps: designing a readout probe for gene coding according to species of a target tissue sample; aiming at the reverse complementary sequences of the target gene set and the read probe group, designing a target probe oligonucleotide pool for targeting a target gene RNA (Ribonucleic Acid) sequence; constructing a targeted probe library for hybridization by using the targeted probe oligonucleotide pool; treating the tissue slice and hybridizing the target probe to prepare an imaging sample; acquiring sequence images of a plurality of rounds of readout probes, capturing RNA original signals, and dyeing the images to obtain original position information of cells in tissues; codes of multiple rounds of imaging signals are combined with a cellular morphology image to be analyzed into an RNA spatial expression matrix of a single cell.

Description

Technical Field

[0001] The present invention relates to a spatial transcriptome detection technology, and in particular to a high-throughput RNA in situ hybridization detection method realized by multi-round imaging combined encoding. Background Art

[0002] Except for viruses, living organisms use single cells as the basic unit of life activities. Single-cell RNA Sequencing (scRNA-seq) technology obtains the genetic information of single cells in biological tissues rather than the mean of tissue genetic information, thereby understanding the genetic heterogeneity of single cells. However, this type of method sacrifices the original position information of cells in tissues.

[0003] Spatial transcriptomics technology can obtain the genetic heterogeneity of single cells while retaining the information of the original position or relative position of the cells. This can not only find heterogeneous cell populations that have been neglected in previous studies, discover important cell type marker genes and regional expression difference marker genes, but also further study the interaction mechanism between cells. It is a powerful tool to help researchers understand important scientific issues such as tissue development, organogenesis, physiological and pathological processes.

[0004] Among the existing spatial transcriptomics detection methods, the methods based on the next generation sequencing (NGS) technology are the most widely used, such as Slide-seq, 10X Visium, Stereo-seq, etc. However, this type of method has many technical limitations: Slide-seq and 10X Visium technologies are difficult to achieve true pre-labeling of single cell spatial barcode information; 10X Visium and Stereo-seq are difficult to optimize a unified permeabilization time for heterogeneous tissues, resulting in large deviations in RNA capture in different spatial regions by these technologies; the RNA capture efficiency of Stereo-seq technology is too low, making it difficult for the final sequencing depth to meet further research needs. Another type of method based on Fluorescence in situ hybridization (FISH) technology can achieve high-resolution RNA in situ signal acquisition, but the limitations of the existing technology are: it is difficult to avoid spatial crowding of fluorescent signals, resulting in signal interference and overlap; because the imaging time is long, it requires high sample processing. Therefore, there is an urgent need in this field for a spatial transcriptome method that can truly achieve efficient and high-throughput single-cell resolution. Summary of the invention

[0005] The present invention aims to solve the above limitations of existing spatial transcriptome technology: 1) Through in situ hybridization imaging of RNA signals, high-throughput spatial transcriptome detection with single-cell or even subcellular resolution is achieved, and indiscriminate detection of different tissue regions is achieved, efficiently capturing RNA information in situ. 2) Through the strategy of multiple rounds of signal combination encoding to dilute the fluorescent signal, combined with the removal of tissue fluorescence background before imaging, the congestion and overlap of RNA signals in tissues are solved. 3) Optimize the sample processing process before imaging to achieve common tissue samples to maintain tissue morphology and RNA stability during long-term imaging signal acquisition.

[0006] In some aspects, provided herein are inventions described in one or more of the following.

[0007] 1. A single-cell spatial transcriptome method based on multi-round imaging encoding, wherein the method comprises the following steps:

[0008] Step S10, designing a readout probe for gene encoding for the species of the target tissue sample, wherein the readout probe is labeled with a fluorescent signal reporter molecule;

[0009] Step S20, designing a targeting probe oligonucleotide pool for targeting the RNA sequence of the target gene according to the reverse complementary sequence of the target gene and the readout probe, wherein the targeting probe comprises the targeting sequence of the target gene and the reverse complementary sequence of the readout probe;

[0010] Step S30, constructing a targeting probe library for hybridization using the targeting probe oligonucleotide pool;

[0011] Step S40, processing tissue sections and hybridizing targeting probes to prepare imaging samples;

[0012] Step S50, collecting sequential imaging signals of the readout probes, wherein the readout probes are hybridized in groups, and a group of readout probes belonging to different fluorescent channels are hybridized for one imaging, thereby collecting multiple rounds of imaging signals, and then collecting cell morphology images;

[0013] Step S60, combining the encoding of multiple rounds of imaging signals with the cell morphology image, and parsing it into an RNA spatial expression matrix of a single cell, wherein the encoding of the target gene is achieved by the combination arrangement of the reverse complementary sequence of the readout probe in the targeting probe.

[0014] 2. The method of claim 1, comprising one or more of the following features:

[0015] 1) The readout probes include more than 2, which are adjusted according to the number of target genes, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 120, 150, 180, 200 or more, for example, between 2 and 200;

[0016] 2) The readout probe is labeled with fluorescent signal reporter molecules for different fluorescent channels;

[0017] 3) Each sequence of the readout probe has no specific binding to the transcriptome sequence of the species of the target sample, and the readout probes have no specific binding to each other.

[0018] 3. The method of claim 1 or 2, comprising one or more of the following features:

[0019] 1) One or both sides of the targeting sequence of the target gene have a spacer sequence, such as one or more thymine deoxyribonucleotides, such as 1 (T), 2 (TT) or 3 (TTT) thymine deoxyribonucleotides;

[0020] 2) The reverse complementary sequence of the readout probe has a spacer sequence on one or both sides;

[0021] 3) the reverse complementary sequence of the readout probe is at the 5' end and / or 3' end of the targeting sequence of the target gene;

[0022] 4) at least a portion of the reverse complementary sequences of all readout probes contained in the targeting probe are arranged in series in the targeting probe;

[0023] 5) The targeting probe comprises PCR amplification primer sequences located at the two ends of the entire sequence, namely, the 5' end primer sequence and the 3' end primer sequence.

[0024] 4. The method of item 3, comprising one or more of the following features:

[0025] 1) The targeting probe comprises an RNA polymerase promoter sequence, which is located at the 5' end of the 3' primer sequence;

[0026] 2) the targeting probe comprises one or more uracil ribonucleotides for primer removal located at the 3' end of the 5' end primer sequence, for example, 1 (U), 2 (UU) or 3 (UUU) uracil ribonucleotides;

[0027] 3) There are spacer sequences on both sides of the targeting sequence of the target gene;

[0028] 4) The reverse complementary sequence of the readout probe has spacer sequences on both sides;

[0029] 5) The targeting probe comprises a PCR amplification primer sequence, a spacer sequence, a reverse complementary sequence of a readout probe, a targeting sequence of a target gene, and an RNA polymerase promoter sequence, optionally wherein one or more spacer sequences are present between the above sequences, and optionally wherein each of the above sequences has a spacer sequence between them.

[0030] 5. The method of any one of items 1-4, wherein multiple targeting probes are designed for each target gene, for example, more than 5, for example, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100 or more, for example, the number can be between 5 and 100, optionally, wherein the readout probe reverse complementary sequences of the multiple targeting probes are the same, while the target gene targeting sequences are different.

[0031] 6. The method of any one of items 1-5, wherein multiple rounds of sequential imaging of readout probes include more than 2 rounds, such as 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50 rounds or more, for example, the number of rounds ranges between 2 and 50 rounds.

[0032] 7. The method of any one of items 1 to 6, wherein step S30 comprises:

[0033] Step S31, using real-time fluorescence quantitative PCR to quantitatively amplify the oligonucleotide pool to obtain a double-stranded DNA nucleic acid pool;

[0034] Step S32, using the double-stranded DNA as a template to perform an in vitro transcription synthesis reaction to obtain an RNA molecule that is reverse complementary to the target gene;

[0035] Step S33, using the above RNA molecule as a template to perform a reverse transcription reaction to obtain a hybrid molecule mixture of the RNA template and the target gene;

[0036] Step S34, removing the 5' end primer from the above mixture;

[0037] Step S35, removing RNA in the hybrid molecule from the above mixture to obtain a targeting probe solution.

[0038] 8. The method of any one of items 1 to 7, wherein step S40 comprises:

[0039] Step S41, slicing the tissue to be processed and permeabilizing the slices;

[0040] Step S42, performing hybridization reaction between the aforementioned synthesized targeting probe and the aforementioned processed tissue section;

[0041] Step S43, clearing the fluorescence background of the hybridized slice.

[0042] 9. The method of any one of items 1 to 8, wherein step S50 comprises:

[0043] The readout probes are hybridized in groups, and a group of readout probes belonging to different fluorescent channels are hybridized for one imaging, non-specific hybridization adsorption is washed off after hybridization, histological staining is performed, and the imaging of this round is collected;

[0044] After the signal collection of the group of readout probes is completed, the group of readout probes is completely removed with an elution solution;

[0045] After one set of readout probe experiments is completed, the next set of readout probe experiments is started, and multiple rounds of readout probe signal acquisition are repeated;

[0046] The tissue sections were stained with dye and images of stained cell morphology were collected.

[0047] 10. The method of any one of items 1 to 9, wherein step S60 comprises:

[0048] By hybridizing the targeted probe of each gene with multiple readout probes, each gene corresponds to a different combination of readout probes, realizing in situ information encoding of the gene. Combined with the single cells segmented from the staining image, the downstream data is finally parsed into a single-cell transcriptome map with spatial information.

[0049] 11. A two-stage probe for high-throughput detection of multi-round encoded in situ information of transcriptome in tissues, wherein the two-stage probe comprises a readout probe and a targeting probe as defined in any one of items 1 to 10.

[0050] 12. A system for implementing the single-cell spatial transcriptome method based on multi-round imaging encoding according to any one of items 1 to 10, the system comprising:

[0051] Microfluidic sample pool,

[0052] A microfluidic liquid control module, in which a set of multi-way valves is connected to the sample pool, and the multi-way valve controls the inflow of multiple rounds of probe solutions connected to the other end.

[0053] Microfluidic gas control module, in which a set of solenoid valves controls the liquid flow rate.

[0054] The imaging module collects signals from different fluorescence channels.

[0055] Automatic liquid flow and imaging control module: The liquid flow control part automatically flows into the sample pool according to the preset order and flow rate sequence to complete the sequence probe hybridization, and at the same time controls the imaging module to automatically collect images of the tissue after each round of hybridization.

[0056] The present invention provides a single-cell spatial transcriptome method based on multi-round imaging encoding, the method comprising:

[0057] Step S10, designing a readout probe set for gene coding for the species of the target tissue sample, wherein the readout probe is a short oligonucleotide sequence of 15 nt to 20 nt, and its sequence has no specific binding to the transcriptome sequence of the species of the target sample, and each end of the readout probe is chemically modified and labeled with a fluorescent signal reporter molecule;

[0058] Step S20, designing a pool of targeting probe oligonucleotides for targeting target gene RNA sequences based on the target gene set and the reverse complementary sequence of the readout probe set;

[0059] A targeting probe oligonucleotide pool is designed for the target gene, and the obtained oligonucleotide pool will be amplified and synthesized in the subsequent step S30 to obtain a targeting probe ultimately used for hybridization reaction.

[0060] The center of each sequence in the oligonucleotide pool is the target gene targeting fragment, and the two sides of the sequence are connected with a thymine deoxyribonucleotide as a space spacer, and the complementary fragments of the read probe sequence in series on both sides are connected. The series read probe sequence can be two or more. A thymine deoxyribonucleotide is also used as a space spacer between the series read probe fragments. The presence of the space spacer reduces the steric hindrance between the read probe, the target probe and the RNA to be tested when the read probe sequence hybridizes, so as to improve the efficiency of the hybridization reaction. The 3' end of the read probe sequence is connected to a T7 promoter sequence for RNA polymerase recognition, and the last two ends of the entire sequence are PCR amplification primer sequences.

[0061] Among them, a thymine deoxyribonucleotide is added to the 3' end of the 5' end primer of the sequence for primer removal involved in the downstream step. The 3' end primer does not need to be removed due to the unique T7 promoter sequence position: in the subsequent step S30, the oligonucleotide pool will be used as a template to amplify double-stranded DNA, and the amplified double-stranded DNA will be used as a template to transcribe and synthesize a large amount of RNA. In this step, since the T7 promoter is located downstream of the 3' end primer of the entire sequence, the T7 promoter sequence and the 3' end primer sequence will not be synthesized, so that there is no need to remove these sequences additionally downstream. If these two sequences exist on the final targeting probe, on the one hand, the length will increase and steric hindrance will be increased, and the hybridization efficiency will be reduced. On the other hand, the non-specific binding of the probe will be increased, and the specificity of the hybridization will be reduced. Therefore, this design is used to improve the hybridization efficiency and the specificity of the hybridization reaction.

[0062] Design 10-40 different sequences for each target gene to increase the signal strength;

[0063] Step S30, constructing a targeting probe library for hybridization using the targeting probe oligonucleotide pool;

[0064] Step S40, processing tissue sections and hybridizing targeting probes to prepare imaging samples;

[0065] Step S50, collecting multiple rounds of sequential imaging of the readout probes, capturing the original RNA signal and obtaining the original position information of the cells in the tissue through DAPI and NISSL staining images;

[0066] Step S60, combining the encoding of multiple rounds of imaging signals with the cell morphology image to analyze the RNA spatial expression matrix of a single cell.

[0067] In some preferred embodiments, step S30 includes:

[0068] Step S31, quantitatively amplifying the oligonucleotide pool by real-time fluorescence quantitative PCR, purifying the amplified product by double-stranded DNA adsorption purification column to obtain a purified double-stranded DNA nucleic acid pool;

[0069] Step S32, using the double-stranded DNA purified above as a template, using T7 RNA polymerase to perform an in vitro transcription synthesis reaction at 37 degrees overnight to obtain an RNA molecule that is reverse complementary to the target fragment;

[0070] Step S33, using the above RNA molecule as a template, and using a primer modified with a uracil ribonucleotide at the 3' end to perform a reverse transcription reaction, to synthesize a hybrid molecule mixture of the RNA template and the target DNA sequence;

[0071] Step S34, treating the mixture with USER enzyme at a ratio of 1:30 to 1:10 to remove the terminal primer;

[0072] Step S35, using a NaOH solution with a final concentration of 0.2-1.0N and an EDTA solution with a final concentration of 0.2-1.0M to perform alkaline hydrolysis at a temperature of 65 to 98 degrees on the RNA in the hybrid molecule. The final product is purified using a single-stranded DNA adsorption purification column to obtain a targeted probe solution.

[0073] In some preferred embodiments, step S40 includes:

[0074] Step S41, cutting the tissue into 5-20 micron tissue slices using a freezing microtome, attaching the slices to the surface of a glass slide treated with D-polylysine, fixing the slices with a PFA solution, and then permeabilizing the slices with 70% ethanol and 8%-10% SDS;

[0075] Step S42, performing hybridization reaction between the synthesized probe solution and the tissue section in a hybridization oven, with the hybridization gradient temperature ranging from 45 degrees to 37 degrees and the hybridization time ranging from 12 hours to 72 hours;

[0076] Step S43, subjecting the protein molecules and RNA molecules in the hybridized slices to an acrylamide group modification reaction, embedding and fixing them in an acrylamide / bisacrylamide cross-linked gel, and using a solution containing proteinase K, Triton-X 100 and SDS diluted 1:50 to 1:200 to clear the fluorescent background of the tissue slices;

[0077] Beneficial effects of the present invention:

[0078] (1) The present invention detects RNA signals based on fluorescence imaging, which can realize the spatial transcriptome signal acquisition of single cells or even subcellular resolution. Cell segmentation is performed by organizing cell morphology images, and the collected RNA signals are attributed to individual cells to obtain single cell spatial transcriptome data.

[0079] (2) The present invention reduces the deviation of signal acquisition in different regions of extremely heterogeneous tissue by removing tissue fluorescence background, and has high sensitivity in acquiring RNA signals in different regions.

[0080] (3) Based on the principle of nucleic acid in situ hybridization, the present invention designs specific targeted nucleic acid probes for the target gene set, thereby improving the capture depth of the target RNA signal.

[0081] (4) The present invention uses a strategy of multiple rounds of fluorescence signal combination encoding to achieve high-throughput RNA signal detection. Diluting the target gene set in multiple groups of fluorescence imaging rounds also solves the problem of optical congestion. In addition, since the tissue fluorescence background is cleared before imaging, the signal-to-noise ratio is increased, thereby further reducing fluorescence signal overlap and congestion.

[0082] (5) In the tissue pretreatment process optimized by the present invention, the tissue to be tested is treated by embedding with hydrogel, so that the RNA signal to be tested and the tissue cells to be tested maintain a long-term stable spatial position, and the degradation of the RNA signal during the detection process is reduced, thereby improving the robustness to ensure stable signal readout during multiple rounds of combined imaging. BRIEF DESCRIPTION OF THE DRAWINGS

[0083] Figure 1 It is a schematic diagram of the process of the high-throughput single-cell spatial transcriptome method based on multiple rounds of imaging encoding of the present invention.

[0084] Figure 2 It is a schematic diagram of the sequence structure of the targeted probe oligonucleotide pool of an embodiment of the high-throughput single-cell spatial transcriptome method based on multiple rounds of imaging encoding of the present invention.

[0085] Figure 3 It is a schematic diagram of the structure of a system for automatically collecting multiple rounds of fluorescence signals from tissues according to the present invention.

[0086] Figure 4 This is an RNA in situ signal acquisition image of an embodiment of the high-throughput single-cell spatial transcriptome method based on multiple rounds of imaging encoding of the present invention (the arrows indicate the RNA molecular signals collected in situ).

[0087] Figure 5 It is an in situ analysis image of the target gene combined with cell morphology information in an embodiment of the high-throughput single-cell spatial transcriptome method based on multiple rounds of imaging encoding of the present invention (shown is the analysis of spatial transcription information in one imaging field of view of mouse embryonic brain tissue, with colors distinguishing different genes. The figure only selects some of the target genes in this embodiment for illustration: Hes5, Sox2, Nkx2-1, Ntrk2, Fam107a, Hopx, Mki67, Ascl1, Dlx2, Dlx5, Dcx, Lgfbp5, Vezf1, Cst3, Pcdh10, Wwtr1, Lxn, Ccn1, Nfia, Dab1, Pdpn, Dlx1, Pbrm1, Sp9, Prox1, Sox11, Epha4, Plxna2, Gad2, Gad1).

[0088] Figure 6 This is an in situ analysis image of cell types in one embodiment of the high-throughput single-cell spatial transcriptome method based on multiple rounds of imaging encoding of the present invention (shown is the cell type information in one imaging field of view of mouse embryonic brain tissue, with colors distinguishing different cell types). DETAILED DESCRIPTION

[0089] The present application will be further described in detail below in conjunction with the accompanying drawings and embodiments. It is to be understood that the specific embodiments described herein are only used to explain the relevant invention, rather than to limit the invention. It should also be noted that, for ease of description, only the parts related to the relevant invention are shown in the accompanying drawings.

[0090] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present application will be described in detail below with reference to the accompanying drawings and in combination with the embodiments.

[0091] The present invention provides a single-cell spatial transcriptome method based on multi-round imaging encoding, the method comprising:

[0092] Step S10, designing a readout probe set for gene encoding according to the species of the target tissue sample;

[0093] Step S20, designing a pool of targeting probe oligonucleotides for targeting target gene RNA sequences based on the target gene set and the reverse complementary sequence of the readout probe set;

[0094] Step S30, constructing a targeting probe library for hybridization using the targeting probe oligonucleotide pool;

[0095] Step S40, processing tissue sections and hybridizing targeting probes to prepare imaging samples;

[0096] Step S50, collecting multiple rounds of sequential imaging of the readout probes, capturing the original RNA signal and obtaining the original position information of the cells in the tissue through DAPI and NISSL staining images;

[0097] Step S60, combining the encoding of multiple rounds of imaging signals with the cell morphology image to analyze the RNA spatial expression matrix of a single cell.

[0098] In order to more clearly illustrate the single-cell spatial transcriptome method based on multiple rounds of imaging encoding of the present invention, Figure 1 Each step in the embodiment of the present invention is described in detail.

[0099] The single-cell spatial transcriptome method based on multi-round imaging encoding in the first embodiment of the present invention includes steps S10 to S60, and each step is described in detail as follows:

[0100] Step S10, designing a readout probe set for gene coding according to the species of the target tissue sample.

[0101] Step S20, designing a pool of targeting probe oligonucleotides for targeting the target gene RNA sequence based on the target gene set and the reverse complementary sequence of the readout probe group.

[0102] A targeting probe oligonucleotide pool is designed for the target gene, and the obtained oligonucleotide pool will be amplified and synthesized in the subsequent step S30 to obtain a targeting probe ultimately used for hybridization reaction.

[0103] The center of each sequence in the oligonucleotide pool is the target gene's targeted fragment, the target gene's targeted fragment has a length between 20nt and 30nt, a GC content between 45% and 65%, and has no off-target after alignment with the genome and transcriptome of the target species.

[0104] The two sides of the target fragment of the target gene are connected with complementary fragments of the readout probe sequence in series with a thymine deoxyribonucleotide as a spacer.

[0105] The tandem readout probe sequences may be two or more, and a thymine deoxyribonucleotide is also used as a spacer between the tandem readout probe fragments.

[0106] The presence of the spacer reduces the steric hindrance between the readout probe, the targeting probe and the RNA to be detected when the readout probe sequence is hybridized, so as to improve the efficiency of the hybridization reaction.

[0107] The 3' end of the readout probe sequence is connected to a T7 promoter sequence for RNA polymerase recognition, and the last two ends of the entire sequence are PCR amplification primer sequences.

[0108] Among them, a thymine deoxyribonucleotide is added to the 3' end of the 5' end primer of the sequence for primer removal involved in the downstream step. The 3' end primer does not need to be removed due to the unique T7 promoter sequence position: in the subsequent step S30, the oligonucleotide pool will be used as a template to amplify double-stranded DNA, and the amplified double-stranded DNA will be used as a template to transcribe and synthesize a large amount of RNA. In this step, since the T7 promoter is located downstream of the 3' end primer of the entire sequence, the T7 promoter sequence and the 3' end primer sequence will not be synthesized, so that there is no need to remove these sequences additionally downstream. If these two sequences exist on the final targeting probe, on the one hand, the length will increase and steric hindrance will be increased, and the hybridization efficiency will be reduced. On the other hand, the non-specific binding of the probe will be increased, and the specificity of the hybridization will be reduced. Therefore, this design is used to improve the hybridization efficiency and the specificity of the hybridization reaction.

[0109] Design 10-40 different sequences for each target gene to increase the signal strength;

[0110] The collection of all targeting probe sequences of all target genes is a targeting probe pool. Some exemplary target genes (taking Hes5, Hopx, and Dlx2 as examples) and exemplary probe sequences designed according to the method of the present invention are shown below:

[0111] Some representative targeted probes of Hes5:

[0112] Hes5 Targeting Probe 1 Sequence:

[0113]

[0114] Hes5 Targeting Probe 2 Sequence:

[0115]

[0116] Hes5 Targeting Probe 3 Sequence:

[0117]

[0118]

[0119] Some representative targeting probes of Hopx: Hopx targeting probe 1 sequence:

[0120]

[0121] Hopx Targeting Probe 2 Sequence:

[0122]

[0123] Hopx Targeting Probe 3 Sequence:

[0124]

[0125] Some representative targeting probes for Dlx2: Sequence of Dlx2 targeting probe 1:

[0126]

[0127] Dlx2 Targeting Probe 2 Sequence:

[0128]

[0129] Dlx2 Targeting Probe 3 Sequence:

[0130]

[0131] Hes5 readout probe: (e.g. the code for the Hes5 gene is a combination of 1-2-3-4)

[0132] Read probe 1 sequence:

[0133] Read probe 2 sequence:

[0134] Read probe 3 sequence:

[0135] Read probe 4 sequence:

[0136] Hopx readout probe: (e.g. the encoding of the Hopx gene is a combination of 5-2-6-7)

[0137] Read probe 5 sequence:

[0138] Read probe 2 sequence:

[0139] Read out probe 6 sequence:

[0140] Read probe 7 sequence:

[0141] Dlx2 readout probe: (e.g. the encoding of the Dlx2 gene is a combination of 8-9-10-7)

[0142] Read out probe 8 sequence:

[0143] Read the probe 9 sequence:

[0144] Read the probe 10 sequence:

[0145] Read probe 7 sequence:

[0146] Step S30, constructing a targeting probe library for hybridization using the targeting probe oligonucleotide pool.

[0147] Step S31, using real-time fluorescence quantitative PCR to quantitatively amplify the oligonucleotide pool (customized and purchased from Twist Bioscience), stopping the synthesis reaction before the plateau phase of the amplification curve is reached, and immediately after the reaction, purifying the amplified product using a double-stranded DNA adsorption purification column, dissolving the product with nuclease-free water to obtain a purified double-stranded DNA nucleic acid pool;

[0148] Step S32, using the double-stranded DNA purified above as a template, using T7 RNA polymerase (NEB, E2050S) at 37 degrees for 18 hours, and performing an in vitro transcription synthesis reaction to obtain an RNA molecule that is reverse complementary to the target fragment and has a ratio of more than 180 times that of the template double-stranded DNA. In this step, the yield of the target sequence is further amplified;

[0149] Step S33, using the above RNA molecule as a template, and using a primer modified with a uracil ribonucleotide at the 3' end to perform a reverse transcription reaction, to synthesize a hybrid molecule mixture of the RNA template and the target DNA sequence;

[0150] Step S34, diluting the mixture with USER enzyme (NEB, M5507) at a ratio of 1:10, and reacting at 37 degrees for 24 hours to remove the 5'-terminal primer modified with uracil ribonucleotide;

[0151] Step S35, using a NaOH solution with a final concentration of 0.25N and an EDTA solution with a final concentration of 0.25M to hydrolyze the RNA in the above hybrid molecule at 95 degrees for more than 15 minutes. Immediately after the reaction, the final product is purified using a single-stranded DNA adsorption purification column to obtain a targeting probe solution, and two volumes of anhydrous ethanol solution and 0.1 volumes of 3M sodium acetate (pH 5.5) solution are added overnight to precipitate the purified single-stranded DNA probe.

[0152] Step S40, processing tissue sections and hybridizing targeting probes to prepare imaging samples.

[0153] Step S41, the tissue to be processed is immediately embedded and stored at -80 degrees when fresh. Before the experiment, the pre-stored tissue is cut into 10-micron thick tissue slices using a freezing microtome, attached to the surface of a glass slide treated with 0.1 mg / mL D-polylysine (Gibco, A3890401) overnight, and the slices are short-fixed with a 4% PFA solution at room temperature for 10 minutes, and the slices are permeabilized with a 70% ethanol solution and an 8% SDS solution respectively;

[0154] Step S42, performing hybridization reaction between the synthesized targeting probe and the treated tissue section in a hybridization oven, with the hybridization gradient temperature ranging from 45 degrees to 37 degrees and the hybridization time being 24 hours;

[0155] Step S43, after the protein molecules and RNA molecules in the hybridized slices are modified with acrylamide groups, they are embedded and fixed in acrylamide / bisacrylamide (A3699, Sigma) cross-linked gel, and the tissue slices are subjected to fluorescence background removal under the condition of overnight reaction at 37 degrees with a solution containing 1:100 diluted proteinase K (NEB, P8102), 0.1% Triton-X 100 (Sigma, 93443) and 1% SDS (Invitrogen, AM9822);

[0156] Step S50, collecting 32 rounds of sequential imaging of the readout probes, capturing the original RNA signal and obtaining the original position information of the cells in the tissue through DAPI and NISSL staining images.

[0157] The readout probes were grouped and hybridized, and a group of readout probes belonging to different fluorescent channels were imaged and hybridized at one time. After hybridization, non-specific hybridization adsorption was eluted with 20% formamide solution (Sigma, F9027), and the cell nucleus was histologically stained with 0.01 mg / mL DAPI (Sigma, D9542) solution. Then, the tissue was immersed in a solution containing 10% D-glucose (Sigma, G5767), 1:100 diluted catalase (Sigma, C3155), 0.5 mg / mL glucose oxidase (SigmaG2133), and 1:1000 diluted RNase inhibitor (Ambion, AM2694), and the imaging of this round was immediately collected. After the signal acquisition of this group of readout probes was completed, the group of readout probes was completely removed with an elution solution containing 60% formamide concentration.

[0158] After one set of readout probe experiments is completed, the next set of readout probe experiments is immediately started, and the process is repeated until 32 rounds of readout probe signal acquisition are completed.

[0159] The 488-channel fluorescently labeled NISSL dye was diluted at a ratio of 1:200, and the tissue sections were stained for 30 minutes at room temperature. The sections were washed with PBST solution for 10 minutes, and the NISSL-stained cell morphology images were immediately collected.

[0160] Step S60, combining the encoding of multiple rounds of imaging signals with the cell morphology image, parsing it into the RNA spatial expression matrix of a single cell, and obtaining information such as the in situ cell type through downstream analysis.

[0161] Through pre-design, the targeted probe of each gene can hybridize multiple readout probes, so each gene corresponds to a different combination of readout probes to achieve in situ information encoding of the gene, and then combined with the single cells segmented from the DAPI staining image taken by the 405 channel, the downstream data is finally parsed into a single-cell transcriptome map with spatial information. In this example, it takes 3 days to stably and automatically collect embryonic mouse brain data of 150 fields of view (field of view area: 210um*210um) for 718 target genes, and 95,425 single cells are obtained. In the end, the number of RNAs measured in each cell is about 100-200.

[0162] The second embodiment of the present invention describes a two-stage probe design scheme for high-throughput detection of multi-round encoding of in situ information of transcriptome in tissues, which includes two parts: a readout probe and a targeting probe, as described in detail as follows:

[0163] Readout probe set: A set of 18nt readout probe oligonucleotide short sequences is designed according to the species of the target tissue, in which each sequence has no specific binding to the species transcriptome sequence of the target sample, and all readout probe oligonucleotide short sequences have no specific binding to each other. The end of each readout probe is chemically modified and connected to a signal reporter molecule labeled with 405, 488, 561, 594, and 647 fluorescent channels.

[0164] Targeting probe pool part: The center of each sequence in the oligonucleotide pool of the targeting probe is a 28nt targeting fragment of the target gene, with a thymine deoxyribonucleotide as a spacer on both sides, connecting the 18nt complementary fragments of the read probe sequence in series on both sides, and a thymine deoxyribonucleotide as a spacer between the series read probe fragments. The 3' end of the sequence is connected to a T7 promoter for RNA polymerase recognition, and the end of the sequence is a 20nt PCR amplification primer sequence at both ends, wherein a thymine deoxyribonucleotide is added to the 3' end of the 5' end primer of the sequence for primer removal involved in the downstream step. Each target gene contains 30 different targeting probe sequences mentioned above, and the reverse complementary sequences of the read probes of these 30 sequences are the same, only the central target gene targeting fragment is different, which is used to multiply the intensity of the signal to improve the sensitivity of the method.

[0165] The encoding of the target gene is achieved by the combination and arrangement of the reverse complementary sequence of the readout probe in the targeting probe: the targeting probe of each gene contains k segments of the reverse complementary sequence of the readout probe arranged in series, and the total number of reverse complementary sequences of the readout probe is n. Then the number of genes that can be encoded C(n,k) is:

[0166]

[0167] This example uses this combination encoding method to achieve high-throughput genome determination using multiple rounds of coding combinations.

[0168] A third embodiment of the present invention includes a system for automatically collecting multiple rounds of fluorescence signals from tissues, the system including the following parts:

[0169] Microfluidic sample pool: It includes a microfluidic pool made of organic glass cut and bonded with a tissue slide. The microfluidic pool made of organic glass cut includes a central probe hybridization pool, liquid flow pipelines on both sides, and liquid inlets and outlets on both sides. The central probe hybridization pool and central probe hybridization pools of different sizes are used to adapt to circular or square tissue areas of different sizes. The liquid flow pipelines on both sides are shorter ones connected to the liquid inlet, and longer ones connected to the liquid outlet. Among them, a bevel angle is added to the indirect part between the central probe hybridization pool and the liquid outlet pipeline, and the top of the entire sample pool is a slope design. The combination of the two allows bubbles in the liquid flow to be smoothly discharged by themselves instead of staying in the central hybridization pool, which solves the interference of tiny bubbles on the hybridization and imaging process. The liquid inlet and outlet on both sides are connected to the liquid path module of the microfluidic control.

[0170] Microfluidic liquid circuit control module: a group of multi-way valves are connected to the sample pool, and the multi-way valves control the inflow of multiple rounds of probe solutions, formamide solutions, DAPI solutions and other solutions connected to the other end.

[0171] Microfluidic gas control module: A group of solenoid valves controls the liquid flow rate. The gas source of the solenoid valve is an air compressor or a nitrogen bottle. The solenoid valve stably controls the pressure range of 0-2000mbar to promote the flow of liquid.

[0172] Imaging module: Signal acquisition is performed in a confocal microscope, including signal sequence acquisition of 405, 488, 561, 594, 647 and other fluorescent channels.

[0173] Automatic liquid flow and imaging control module: The liquid flow control part automatically flows into the sample pool according to the preset order and flow rate sequence to complete the sequence probe hybridization, and at the same time controls the imaging module to automatically collect images of the tissue after each round of hybridization.

[0174] It should be noted that the above embodiment provides a system for automatically collecting multiple rounds of fluorescence signals from tissues, and only uses the division of the above functional modules as an example. In practical applications, the above functions can be assigned to different functional modules as needed, that is, the modules or steps in the embodiments of the present invention can be decomposed or combined. For example, the modules in the above embodiments can be combined into one module, or further divided into multiple sub-modules to complete all or part of the functions described above. The names of the modules and steps involved in the embodiments of the present invention are only for distinguishing the modules or steps, and are not regarded as improper limitations of the present invention.

[0175] So far, the technical solutions of the present invention have been described in conjunction with the preferred embodiments shown in the accompanying drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A single-cell spatial transcriptome method based on multi-round imaging encoding, wherein the method comprises the following steps: Step S10, designing a readout probe for gene encoding for the species of the target tissue sample, wherein the readout probe is labeled with a fluorescent signal reporter molecule; Step S20, designing a targeting probe oligonucleotide pool for targeting the RNA sequence of the target gene according to the reverse complementary sequence of the target gene and the readout probe, wherein the targeting probe comprises the targeting sequence of the target gene and the reverse complementary sequence of the readout probe; Step S30, constructing a targeting probe library for hybridization using the targeting probe oligonucleotide pool; Step S40, processing tissue sections and hybridizing targeting probes to prepare imaging samples; Step S50, collecting sequential imaging signals of the readout probes, wherein the readout probes are hybridized in groups, and a group of readout probes belonging to different fluorescent channels are hybridized for one imaging, thereby collecting multiple rounds of imaging signals, and then collecting cell morphology images; Step S60, combining the encoding of multiple rounds of imaging signals with the cell morphology image, and parsing it into an RNA spatial expression matrix of a single cell, wherein the encoding of the target gene is achieved by the combination arrangement of the reverse complementary sequence of the readout probe in the targeting probe.

2. The method of claim 1, comprising one or more of the following features: 1) The readout probes include more than 2, which are adjusted according to the number of target genes, for example, between 2 and 200; 2) The readout probe is labeled with fluorescent signal reporter molecules for different fluorescent channels; 3) Each sequence of the readout probe has no specific binding to the transcriptome sequence of the species of the target sample, and the readout probes have no specific binding to each other.

3. The method of claim 1 or 2, comprising one or more of the following features: 1) One or both sides of the targeting sequence of the target gene have a spacer sequence, such as one or more thymine deoxyribonucleotides, such as 1 (T), 2 (TT) or 3 (TTT) thymine deoxyribonucleotides; 2) The reverse complementary sequence of the readout probe has a spacer sequence on one or both sides; 3) the reverse complementary sequence of the readout probe is at the 5' end and / or 3' end of the targeting sequence of the target gene; 4) at least a portion of the reverse complementary sequences of all readout probes contained in the targeting probe are arranged in series in the targeting probe; 5) The targeting probe comprises PCR amplification primer sequences located at the two ends of the entire sequence, namely, the 5' end primer sequence and the 3' end primer sequence.

4. The method of claim 3, comprising one or more of the following features: 1) The targeting probe comprises an RNA polymerase promoter sequence, which is located at the 5' end of the 3' primer sequence; 2) the targeting probe comprises one or more uracil ribonucleotides for primer removal located at the 3' end of the 5' end primer sequence, for example, 1 (U), 2 (UU) or 3 (UUU) uracil ribonucleotides; 3) There are spacer sequences on both sides of the targeting sequence of the target gene; 4) The reverse complementary sequence of the readout probe has spacer sequences on both sides; 5) The targeting probe comprises a PCR amplification primer sequence, a spacer sequence, a reverse complementary sequence of a readout probe, a targeting sequence of a target gene, and an RNA polymerase promoter sequence, optionally wherein one or more spacer sequences are present between the above sequences, and optionally wherein each of the above sequences has a spacer sequence between them.

5. The method of any one of claims 1 to 4, wherein multiple targeting probes are designed for each target gene, for example, more than 5, and the number can be between 5 and 100, and optionally, the readout probe reverse complementary sequences of the multiple targeting probes are the same, while the target gene targeting sequences are different.

6. The method of any one of claims 1 to 5, wherein multiple rounds of sequential imaging of readout probes comprise more than 2 rounds, such as the number of rounds ranges between 2 and 50 rounds.

7. The method of any one of claims 1 to 6, wherein step S30 comprises: Step S31, using real-time fluorescence quantitative PCR to quantitatively amplify the oligonucleotide pool to obtain a double-stranded DNA nucleic acid pool; Step S32, using the double-stranded DNA as a template to perform an in vitro transcription synthesis reaction to obtain an RNA molecule that is reverse complementary to the target gene; Step S33, using the above RNA molecule as a template to perform a reverse transcription reaction to obtain a hybrid molecule mixture of the RNA template and the target gene; Step S34, removing the 5' end primer from the above mixture; Step S35, removing RNA in the hybrid molecule from the above mixture to obtain a targeting probe solution.

8. The method of any one of claims 1 to 7, wherein step S40 comprises: Step S41, slicing the tissue to be processed and permeabilizing the slices; Step S42, performing hybridization reaction between the aforementioned synthesized targeting probe and the aforementioned processed tissue section; Step S43, clearing the fluorescence background of the hybridized slice.

9. The method of any one of claims 1 to 8, wherein step S50 comprises: The readout probes are hybridized in groups, and a group of readout probes belonging to different fluorescent channels are hybridized for one imaging, non-specific hybridization adsorption is washed off after hybridization, histological staining is performed, and the imaging of this round is collected; After the signal acquisition of the group of readout probes is completed, the group of readout probes is completely removed by using an elution solution; After one set of readout probe experiments is completed, the next set of readout probe experiments is started, and multiple rounds of readout probe signal acquisition are repeated until completion; The tissue sections were stained with dye and images of stained cell morphology were collected.

10. The method of any one of claims 1 to 9, wherein step S60 comprises: By hybridizing the targeted probe of each gene with multiple readout probes, each gene corresponds to a different combination of readout probes, realizing in situ information encoding of the gene. Combined with the single cells segmented from the staining image, the downstream data is finally parsed into a single-cell transcriptome map with spatial information.

11. A two-stage probe for high-throughput detection of multi-round encoded in situ information of transcriptome in tissues, wherein the two-stage probe comprises a readout probe and a targeting probe as defined in any one of claims 1 to 10.

12. A system for implementing the single-cell spatial transcriptome method based on multi-round imaging encoding according to any one of claims 1 to 10, the system comprising: Microfluidic sample pool, A microfluidic liquid control module, in which a set of multi-way valves is connected to the sample pool, and the multi-way valve controls the inflow of multiple rounds of probe solutions connected to the other end. Microfluidic gas control module, in which a set of solenoid valves controls the liquid flow rate. The imaging module collects signals from different fluorescence channels. Automatic liquid flow and imaging control module: The liquid flow control part automatically flows into the sample pool according to the preset order and flow rate sequence to complete the sequence probe hybridization, and at the same time controls the imaging module to automatically collect images of the tissue after each round of hybridization.

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