CN117385477A - Chip for space transcriptome sequencing, preparation method thereof and space transcriptome sequencing method - Google Patents

Chip for space transcriptome sequencing, preparation method thereof and space transcriptome sequencing method Download PDF

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CN117385477A
CN117385477A CN202311573159.8A CN202311573159A CN117385477A CN 117385477 A CN117385477 A CN 117385477A CN 202311573159 A CN202311573159 A CN 202311573159A CN 117385477 A CN117385477 A CN 117385477A
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廖人杰
刘二凯
陈爽
赵陆洋
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Shenzhen Sailu Medical Technology Co ltd
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Abstract

The invention provides a preparation method of a chip. The method comprises the following steps: ligating the targeting probe and the poly-T probe to a chip substrate comprising a plurality of oligonucleotide sequences to obtain the chip; wherein at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound. The preparation method is simple, and the prepared chip not only can comprehensively capture mRNA containing poly-A tail, but also can capture the nucleotide sequence of specific target molecules, thereby realizing targeted and non-targeted capture.

Description

Chip for space transcriptome sequencing, preparation method thereof and space transcriptome sequencing method
Technical Field
The invention belongs to the technical field of gene sequencing, and particularly relates to a chip for space transcriptome sequencing, a preparation method thereof and a space transcriptome sequencing method.
Background
In recent years, mainly developed countries (e.g., united states, united kingdom, france, etc.) have continued to use genetic technology as a national strategy, while space histology is more a hotspot of current research. Space histology provides important bioinformatic support for cell typing, cell state characterization, cell-cell interactions through in situ characterization of tissue transcriptomes. Has wide application in the fields of biomedicine such as neuroscience, tumor microenvironment research, immunology, disease marker searching and the like. The current space histology technology mainly captures mRNA molecules carrying poly-A tail by using poly-T sequence to realize comprehensive characterization of space transcriptome, but has a certain limitation on capturing efficiency of mRNA molecules, which can cause loss of some target molecules.
Therefore, there is a need to develop a spatial transcriptomics chip and method that can achieve both targeted and non-targeted capture.
Disclosure of Invention
The present invention aims to solve at least one of the technical problems existing in the prior art to at least some extent. Therefore, the invention provides a space transcriptomics chip capable of realizing targeted and non-targeted capturing at the same time, and the space transcriptomics chip can realize targeted and non-targeted capturing at the same time, in particular to capture RNA without poly-A tail.
The present invention has been completed based on the following findings by the inventors:
traditional space transcriptomics technology mainly adopts a poly-T sequence to capture mRNA molecules carrying a poly-A tail, so that comprehensive characterization of space transcriptomes is realized. However, in the conventional space transcriptomics technology, since mRNA is very easily degraded in the transcription process of a sample and is limited by a tissue processing mode, the capturing efficiency of the mRNA molecule is limited, which may cause the loss of an important target molecule. Moreover, for mRNA without poly-a tail (e.g., prokaryotic mRNA (bacterial mRNA), other RNAs (e.g., rRNA, lncRNA), etc.), traditional spatial transcriptomics techniques cannot capture its mRNA for characterization, which makes intestinal-microbial spatial profiling difficult in related studies of intestinal microorganisms.
However, the invention is based on the second generation sequencing technology, a DNA lattice (or oligonucleotide cluster) carrying space coding sequences is formed on the surface of a sequencing chip by bridge amplification, and then a complementary sequence probe and a poly-T probe of a target molecule are connected at the same time, so that the target capture of the specific target molecule can be realized while the non-target comprehensive capture of mRNA can be realized.
Based on this, in a first aspect of the invention, the invention proposes a chip. According to an embodiment of the invention, the chip comprises: chip substrate, targeting probe and poly-T probe; wherein the chip substrate comprises a plurality of oligonucleotide sequences, and each of the plurality of oligonucleotide sequences is independently connected with the targeting probe and the poly-T probe; at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound. The chip of the invention not only can comprehensively capture mRNA containing poly-A tail, but also can realize targeted and non-targeted capture on the nucleotide sequence of specific target molecules.
According to an embodiment of the invention, the gene sequence to be bound comprises at least one of DNA, lncRNA, mRNA and rRNA.
According to an embodiment of the invention, the gene sequence to be bound is RNA without a poly-A tail.
According to an embodiment of the present invention, the ratio of the number of the targeting probes to the poly-T probes on the chip substrate is (0.1-2): (1-2).
According to an embodiment of the present invention, the ratio of the number of the targeting probes to the poly-T probes on the chip substrate is (0.1 to 0.2): (1-2).
According to an embodiment of the present invention, the ratio of the number of the targeting probes to the poly-T probes on the chip substrate is (0.2-2): (1-2).
According to an embodiment of the invention, the targeting probe and/or poly-T probe is linked to the oligonucleotide sequence via a phosphodiester linkage.
According to an embodiment of the present invention, the oligonucleotide sequence comprises, in order from the 5 'end to the 3' end, a linker sequence 1, a sequencing primer, a spatial coding sequence sequencing primer and a linker sequence 2.
According to an embodiment of the invention, the 5 'end of the targeting probe and/or poly-T probe is attached to the 3' end of the linker sequence 2.
According to an embodiment of the invention, the 5' end of the linker sequence 1 is attached to the chip substrate.
In a second aspect of the invention, the invention provides a method of preparing a chip. According to an embodiment of the invention, the method comprises: ligating the targeting probe and the poly-T probe to a chip substrate comprising a plurality of oligonucleotide sequences to obtain the chip; wherein at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound. The preparation method is simple, and the prepared chip not only can comprehensively capture mRNA containing poly-A tail, but also can realize targeted and non-targeted capture on the nucleotide sequence of a specific target molecule.
According to an embodiment of the invention, the gene sequence to be bound comprises at least one of DNA, lncRNA, mRNA and rRNA.
According to an embodiment of the invention, the gene sequence to be bound is RNA without a poly-A tail.
According to an embodiment of the present invention, the oligonucleotide sequence comprises, in order from the 5 'end to the 3' end, a linker sequence 1, a sequencing primer, a spatial coding sequence sequencing primer and a linker sequence 2.
According to an embodiment of the present invention, the plurality of oligonucleotide sequences are obtained after a PCR amplification process in which the adaptor sequence 1 is used as a primer and the complementary strand of the oligonucleotide sequence is used as a template.
According to an embodiment of the present invention, after the PCR amplification process and before the ligation process, the oligonucleotide sequences are subjected to spatial coding sequencing in advance to obtain spatial coordinates of each oligonucleotide sequence of the chip substrate containing the plurality of oligonucleotide sequences.
According to an embodiment of the present invention, the connection processing is performed by: mixing a guide sequence, a polymerase or ligase, the targeting probe and a poly-T probe with the chip substrate comprising a plurality of oligonucleotide sequences; subjecting the mixed reaction product to denaturation treatment so as to obtain the chip.
According to an embodiment of the invention, the mixing reaction is carried out at 35-38 ℃ for 0.5-12 h.
According to an embodiment of the present invention, the polymerase or ligase is added in an amount of 3 to 5U/. Mu.L.
According to an embodiment of the present invention, the molar ratio of the guide sequence, the targeting probe and the poly-T probe is (1-2): (0.1-2): (1-2).
According to an embodiment of the invention, the guide sequence comprises a complementary sequence 1 and a linking sequence 1, at least part of the complementary sequence 1 being complementary paired with at least part of the linker sequence 2, at least part of the linking sequence 1 being complementary paired with at least part of the targeting probe and/or poly-T probe.
According to an embodiment of the invention, the targeting probe and/or poly-T probe comprises a linker sequence 2 and a targeting sequence and/or poly-T sequence, the 3 'end of the linker sequence 2 being linked to the 5' end of the targeting sequence and/or poly-T sequence, at least part of the linker sequence 2 being complementarily paired with at least part of the linker sequence 1.
In a third aspect of the invention, the invention provides a method of spatial transcriptomic sequencing. According to an embodiment of the invention, the method comprises: spatial transcriptomic sequencing of the tissue sample to be tested is performed using the chip according to the first aspect or the chip obtained according to the method according to the second aspect. It will be appreciated that the chip according to the first aspect or the chip obtained according to the second aspect may be used to capture the entire range of mRNA containing poly-A tail and the gene sequence of a specific target molecule, and thus, both targeted and non-targeted capture, especially of target molecules (e.g.rare cell type markers, bacteria in tissue samples) may be achieved by the method of the invention, by means of the targeting probe to break through the limitations of poly-T capture efficiency.
According to the embodiment of the invention, the tissue sample to be detected is subjected to slicing treatment in advance.
According to an embodiment of the invention, the spatial transcriptomic sequencing is performed by: contacting the tissue sample to be tested with a targeting probe and/or a poly-T probe on the chip; sequentially carrying out tissue fixation treatment and tissue permeabilization treatment on the contact treatment product; sequentially performing reverse transcription treatment and RNA denaturation removal treatment on the tissue permeabilization treatment product; sequentially carrying out second chain extension reaction and elution treatment on the RNA denaturation removal treatment product so as to obtain a second chain, wherein the second chain sequence comprises information of a gene sequence to be combined; carrying out library building and sequencing treatment on the second chain; and analyzing the data obtained by the library construction and sequencing treatment so as to obtain a spatial transcription map of a sample to be detected or a spatial distribution map of a host of the gene sequence to be combined.
Additional aspects and advantages of the invention will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the invention.
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The foregoing and/or additional aspects and advantages of the invention will become apparent and may be better understood from the following description of embodiments taken in conjunction with the accompanying drawings in which:
FIG. 1 is a schematic diagram of the chip preparation in example 1 of the present invention;
FIG. 2 is a schematic representation of a seed library of capture probes of the present invention;
FIG. 3 is a schematic diagram showing the capturing, pooling and sequencing of mRNA in example 1 of the present invention;
FIG. 4 is a graph showing the capture heat of the mRNA molecules of the transcription map of the small intestine space in example 1 of the present invention;
FIG. 5 is a graph showing the abundance distribution of a portion of bacteria in a section of small intestine in example 1 of the present invention.
Detailed Description
Embodiments of the present invention are described in detail below. The following examples are illustrative only and are not to be construed as limiting the invention.
It should be noted that the terms "first," "second," and "second" are used for descriptive purposes only and are not to be construed as indicating or implying a relative importance or implying a number of technical features being indicated. Thus, a feature defining "a first" or "a second" may explicitly or implicitly include one or more such feature. Further, in the description of the present invention, unless otherwise indicated, the meaning of "a plurality" is two or more.
In this document, the terms "comprise" or "include" are used in an open-ended fashion, i.e., to include what is indicated by the present invention, but not to exclude other aspects.
In this document, the terms "optionally," "optional," or "optionally" generally refer to the subsequently described event or condition may, but need not, occur, and the description includes instances in which the event or condition occurs, as well as instances in which the event or condition does not.
The invention provides a chip, a preparation method thereof and a space transcriptomics sequencing method, and the chip and the preparation method are respectively described in detail below.
Chip
In a first aspect of the invention, the invention proposes a chip. According to an embodiment of the invention, the chip comprises: chip substrate, targeting probe and poly-T probe; wherein the chip substrate comprises a plurality of oligonucleotide sequences, and each of the plurality of oligonucleotide sequences is independently connected with the targeting probe and the poly-T probe; at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound. The chip of the invention not only can comprehensively capture mRNA containing poly-A tail, but also can realize targeted and non-targeted capture on the nucleotide sequence of specific target molecules.
As used herein, the term "gene sequence to be bound" refers to the sequence of a target molecule to be bound, i.e., a molecule of interest to the user, such as a rare cell type marker, a genomic target region sequence, an RNA sequence (DNA, lncRNA sequence, mRNA sequence or rRNA sequence) of other species (e.g., bacteria), and the like.
According to an embodiment of the invention, the gene sequences to be bound include, but are not limited to, at least one of lncRNA, mRNA and rRNA.
According to an embodiment of the invention, the gene sequence to be bound is RNA without a poly-A tail.
Illustratively, the gene sequence to be bound may be a bacterial 16S rRNA sequence. Therefore, the chip can realize construction of a host space transcriptome and simultaneously characterize the species and space position of bacteria in host species, so that research on the interaction of host and microorganism is possible.
Illustratively, the targeting probe may be a bacterial 16S rRNA probe.
Illustratively, the targeting probe may be a targeting probe that binds lncRNA.
Illustratively, the targeting probe may be a targeting probe that binds bacterial mRNA.
According to an embodiment of the present invention, the ratio of the number of the targeting probes to the poly-T probes on the chip substrate is (0.1-2): (1-2), for example, (0.1-1.5): (1-2) and (0.1-1): (1-2) and (0.1-0.5): (1-2) and (0.1-0.2): (1-2) and (0.2-1.5): (1-2) and (0.2-1.0): (1-2) and (0.2-0.5): (1-2) and (0.1-2): (1-1.5) and (0.1-2): (1.5 to 2), preferably (0.1 to 1): 1. experiments show that the adoption of the ratio can further improve the targeted and non-targeted capturing efficiency, avoid the low capturing efficiency of the targeted probe or avoid the low capturing efficiency of mRNA by the poly-T probe.
As used herein, the term "quantitative ratio" refers to the ratio of the number of oligonucleotide sequences attached to a targeting probe to the number of oligonucleotide sequences attached to a poly-T probe on a chip substrate.
According to an embodiment of the present invention, the ratio of the number of the targeting probes to the poly-T probes on the chip substrate is (0.1 to 0.2): (1-2).
According to an embodiment of the present invention, the ratio of the number of the targeting probes to the poly-T probes on the chip substrate is (0.2-2): (1-2).
According to an embodiment of the present invention, the chip substrate includes a plurality of oligonucleotide clusters, and each oligonucleotide cluster includes a plurality of oligonucleotide sequences. The number of the targeting probes or the poly-T probes in each oligonucleotide cluster is almost the same, namely, each oligonucleotide cluster contains the same number of the targeting probes and the same number of the poly-T probes, and the number ratio of the targeting probes to the poly-T probes in each oligonucleotide cluster is (0.1-2): (1-2).
According to an embodiment of the invention, the targeting probe and/or poly-T probe is linked to the oligonucleotide sequence via a phosphodiester linkage.
According to an embodiment of the present invention, the oligonucleotide sequence comprises, in order from the 5 'end to the 3' end, a linker sequence 1, a sequencing primer, a spatial coding sequence sequencing primer and a linker sequence 2.
The plurality of oligonucleotide sequences are obtained by a PCR amplification process in which the adaptor sequence 1 is used as a primer and the complementary strand of the oligonucleotide sequence is used as a template. Before the oligonucleotide sequences are not linked, two linker sequences are present on the chip substrate, the complementary sequence of linker sequence 2 and linker sequence 1, respectively; bridge amplification can be performed by the complementary sequence of the adaptor sequence 2 of the oligonucleotide sequence and the adaptor sequence 2 on the chip substrate. The linker sequence 1 and the linker sequence 2 of the present invention may be selected according to practical conditions, and specific sequences are not limited as long as they can satisfy bridge amplification on a chip substrate.
Illustratively, the oligonucleotide sequence is CAAGCAGAAGACGGCATACGAGATTCTTTCCCTACACGACGCTCTTCCGATCTNNNNN NNNNNNNNNNNNNNNNNNNNNNNNNCCTCCCACCCTCGCTTGCCCTGCCTCCAATG GAGATATAATACTCCGGCATTGTGTG (SEQ ID NO: 1).
Illustratively, the linker sequence 1 is CAAGCAGAAGACGGCATACGAGAT (SEQ ID NO: 2); the linker sequence 2 was GGAGATATAATACTCCGGCATTGTGTG (SEQ ID NO: 3).
In an alternative embodiment of the present invention, the sequencing primer is used for a sequencing primer used in a subsequent library-building and sequencing process step, and the specific sequence is not limited as long as the sequencing can be performed in the subsequent library-building and sequencing process step.
In an alternative embodiment of the invention, the spatially encoded sequences (also known as Barcode) are used to label different oligonucleotide sequences on the chip substrate; spatially encoded sequence sequencing primers are used to sequence spatially encoded sequences by primer hybridization and spatially localize each spatially encoded sequence based on the image to obtain spatial coordinates. The specific sequences of the spatial coding sequences and the spatial coding sequence sequencing primers are not limited as long as spatial localization can be achieved.
According to an embodiment of the invention, the 5 'end of the targeting probe and/or poly-T probe is attached to the 3' end of the linker sequence 2.
According to an embodiment of the invention, the 5' end of the linker sequence 1 is attached to the chip substrate.
In some alternative embodiments of the invention, the 5' end of the linker sequence 1 and the chip substrate are connected by a chemical bond.
Illustratively, the 5 'end of the linker sequence 1 has a DBCO (dibenzocyclooctyne) modification and the chip substrate has an azide modification, the 5' end of the linker sequence 1 and the chip substrate being connected by a Click Chemistry (Click Chemistry) reaction of DBCO and azide.
Method for preparing chip
In a second aspect of the invention, the invention provides a method of preparing a chip. According to an embodiment of the invention, the method comprises: ligating the targeting probe and the poly-T probe to a chip substrate comprising a plurality of oligonucleotide sequences to obtain the chip; wherein at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound. The method can prepare the space transcriptomics chip capable of realizing targeted capture and non-targeted capture simultaneously, and the preparation method is simple.
As used herein, "ligation" refers to any reaction or treatment in which a targeting probe and a poly-T probe are ligated to a chip substrate containing a plurality of oligonucleotide sequences, and is specifically not limited and is within the scope of the present invention. Illustratively, the ligation process may be a ligase reaction or a polymerase extension reaction to generate targeting probes or poly-T probes at the ends of the oligonucleotide sequences on the chip substrate.
According to an embodiment of the invention, the gene sequences to be bound include, but are not limited to, at least one of DNA, lncRNA, mRNA and rRNA.
According to an embodiment of the invention, the gene sequence to be bound is RNA without a poly-A tail.
Illustratively, the gene sequence to be bound may be a bacterial 16S rRNA sequence.
Illustratively, the targeting probe may be a bacterial 16S rRNA probe.
Illustratively, the targeting probe may be a targeting probe that binds lncRNA.
Illustratively, the targeting probe may be a targeting probe that binds bacterial mRNA.
According to an embodiment of the present invention, the oligonucleotide sequence comprises, in order from the 5 'end to the 3' end, a linker sequence 1, a sequencing primer, a spatial coding sequence sequencing primer and a linker sequence 2.
According to an embodiment of the present invention, the plurality of oligonucleotide sequences are obtained after a PCR amplification process in which the adaptor sequence 1 is used as a primer and the complementary strand of the oligonucleotide sequence is used as a template.
It should be noted that, before the oligonucleotide sequences are not linked, two linker sequences are present on the chip substrate, the complementary sequence of linker sequence 2 and linker sequence 1, respectively; the complementary strand of the linker sequence 1 in the complementary strand of the oligonucleotide sequence is complementarily paired with the linker sequence 1 on the chip substrate, and then PCR amplification is performed by using the linker sequence 1 as a primer and the complementary strand of the oligonucleotide sequence as a template, so that individual oligonucleotide clusters are grown on the chip substrate, each oligonucleotide cluster comprising a plurality of oligonucleotide sequences of identical sequence.
Illustratively, the PCR amplification is bridge amplification (or bridge PCR amplification).
According to an embodiment of the present invention, after the PCR amplification process and before the ligation process, the oligonucleotide sequences are subjected to spatial coding sequencing in advance to obtain spatial coordinates of each oligonucleotide sequence of the chip substrate containing the plurality of oligonucleotide sequences. Thus, the spatial coding sequences are sequenced by primer hybridization with the addition of primers that can be complementarily paired with the spatial coding sequence sequencing primers, and each spatial coding sequence is spatially located from the image to obtain spatial coordinates.
According to an embodiment of the present invention, the connection processing is performed by: mixing a guide sequence, a ligase or a polymerase, the targeting probe and a poly-T probe with the chip substrate containing the plurality of oligonucleotide sequences; subjecting the mixed reaction product to denaturation treatment so as to obtain the chip.
According to an embodiment of the invention, the mixing reaction is carried out at 35-38 ℃ for 0.5-12 h.
According to an embodiment of the present invention, the temperature of the mixing reaction is 35 ℃, 36 ℃,37 ℃, 38 ℃; the mixing reaction time is 0.5-10 h, 0.5-5 h, 0.5-4 h, 0.5-3 h, 0.5-2 h, 0.5-1 h, 1-12 h, 5-12 h and 6-12 h.
According to an embodiment of the present invention, the ligase or polymerase is added in an amount of 3 to 5U/. Mu.L, such as 3U/. Mu.L, 4U/. Mu.L, 5U/. Mu.L or any two thereof as a range value between the end values.
In some alternative embodiments of the invention, the ligase is a T4 ligase.
In some alternative embodiments of the invention, the T4 ligase is added in an amount of 3 to 5U/. Mu.L.
In some alternative embodiments of the invention, the polymerase is a DNA polymerase.
According to an embodiment of the present invention, the molar ratio of the guide sequence, the targeting probe and the poly-T probe is (1-2): (0.1-2): (1-2). The prepared chip not only can simultaneously capture mRNA containing poly-A tail and the gene sequence of a specific target molecule, but also can ensure the acquisition quantity of the gene sequence of the specific target molecule.
According to an embodiment of the present invention, the molar ratio of the guide sequence, the targeting probe and the poly-T probe is (1-2): (0.1-0.2): (1-2).
According to an embodiment of the present invention, the molar ratio of the guide sequence, the targeting probe and the poly-T probe is (1-2): (0.2-2): (1-2).
According to an embodiment of the present invention, the molar ratio of the guide sequence, the targeting probe and the poly-T probe is (1-2): (0.1-1): (1-2).
According to an embodiment of the invention, the guide sequence comprises a complementary sequence 1 and a linking sequence 1, at least part of the complementary sequence 1 being complementary paired with at least part of the linker sequence 2, at least part of the linking sequence 1 being complementary paired with at least part of the targeting probe and/or poly-T probe.
According to an embodiment of the invention, the targeting probe and/or poly-T probe comprises a linker sequence 2 and a targeting sequence and/or poly-T sequence, the 3 'end of the linker sequence 2 being linked to the 5' end of the targeting sequence and/or poly-T sequence, at least part of the linker sequence 2 being complementarily paired with at least part of the linker sequence 1. The targeting probe comprises a linker sequence 2 and a targeting sequence, the poly-T probe comprises a linker sequence 2 and a poly-T sequence, whereby the targeting probe, the poly-T probe, the targeting sequence and a DNA polymerase or ligase (e.g.T4ligase) are added simultaneously, at least part of the complementary sequence 1 of the targeting sequence is complementarily paired with at least part of the linker sequence 2, and then the targeting probe and/or the poly-T probe is located at the 3' end of the linker sequence 2 by complementation of at least part of the linker sequence 2 and at least part of the linker sequence 1, and then the 5' end of the linker sequence 2 is connected to the 3' end of the linker sequence 2 by a DNA polymerase or ligase (e.g.T4ligase).
It is noted that at least part of the targeting sequence and at least the complementary pair of the gene sequence to be bound, the poly-T sequence and the poly-A complementary pair.
According to an embodiment of the invention, the 5' end of the targeting probe and/or poly-T probe has a phosphorylation modification prior to the ligation process.
Spatial transcriptomics sequencing method
In a third aspect of the invention, the invention provides a method of spatial transcriptomic sequencing. According to an embodiment of the invention, the method comprises: spatial transcriptomic sequencing of the tissue sample to be tested is performed using the chip according to the first aspect or the chip obtained according to the method according to the second aspect. It will be appreciated that the chip according to the first aspect or the chip obtained according to the second aspect may be used to capture the entire range of mRNA containing poly-A tail and the nucleotide sequence of a specific target molecule, and thus, both targeted and non-targeted capture, especially of target molecules (e.g.rare cell type markers, bacteria in tissue samples) may be achieved using the methods of the invention, by means of targeting probes to break through the limitations of poly-T capture efficiency.
According to the embodiment of the invention, the tissue sample to be detected is subjected to slicing treatment in advance.
According to an embodiment of the present invention, as shown in fig. 3, the spatial transcriptomic sequencing is performed by: contacting the tissue sample to be tested with a targeting probe and/or a poly-T probe on the chip; sequentially carrying out tissue fixation treatment and tissue permeabilization treatment on the contact treatment product; sequentially performing reverse transcription treatment and RNA denaturation removal treatment on the tissue permeabilization treatment product; sequentially carrying out second chain extension reaction and elution treatment on the RNA denaturation removal treatment product so as to obtain a second chain, wherein the second chain sequence comprises information of a gene sequence to be combined; carrying out library building and sequencing treatment on the second chain; and analyzing the data obtained by the library construction and sequencing treatment so as to obtain a spatial transcription map of a sample to be detected or a spatial distribution map of a nucleotide sequence host to be combined.
In the text, "tissue fixation treatment" and "tissue curing treatment" are synonymous; "RNA digestion treatment" is synonymous with "RNA denaturation removal treatment".
According to an embodiment of the present invention, the tissue fixation treatment is performed in methanol at (-20 to-10) deg.C for 20 to 40 minutes.
According to an embodiment of the invention, the tissue permeabilization treatment is performed with pepsin.
In an alternative embodiment of the invention, the tissue permeabilization process comprises the steps of: pepsin is adopted to treat the tissue fixation treatment product for 5 to 20 minutes at the temperature of between 35 and 38 ℃. Pepsin is provided in the form of a pepsin solution, the concentration of pepsin in the pepsin solution being 1mg/mL.
According to an embodiment of the invention, the tissue permeabilization treatment is performed sequentially with lysozyme and pepsin.
Illustratively, the tissue permeabilization process comprises the steps of: 1) Treating the tissue fixation treatment product for 0.5-2 h under the room temperature condition by adopting 150-250 mug/mu L lysozyme; 2) And (3) treating the chip subjected to lysozyme treatment by pepsin at the temperature of 35-38 ℃ for 5-20 min. Pepsin is provided in the form of a pepsin solution, the concentration of pepsin in the pepsin solution being 1mg/mL.
According to an embodiment of the present invention, the reverse transcription process is performed by a reverse transcription mixing reagent; the reverse transcription mix included 2. Mu.L of Maxima H-Rtase reverse transcriptase, 1. Mu.L of RNase inhibitor (RNase inhibitor), 8. Mu.L of 5xRT Buffer, 4. Mu.L of 10mM dNTP mix, 8. Mu.L of 20% polysucrose (Ficoll PM-400), 4mL of actinomycin D (Actinomycin D), 13. Mu.L of nuclease free water (Nuclease free water).
According to an embodiment of the present invention, the reverse transcription treatment product and the exonuclease are mixed and reacted at 35 to 38℃for 30 to 60 minutes before the RNA denaturation removal treatment.
According to an embodiment of the invention, the RNA denaturation removal treatment is performed using potassium hydroxide solution.
In an alternative embodiment of the invention, the concentration of the potassium hydroxide solution is 70 to 90mM.
According to an embodiment of the invention, the second strand extension reaction is performed in a second strand synthesis mixture comprising 3. Mu.L Klenow fragment, 3. Mu.L NEB buffer, 3. Mu.L 10mM dNTP mix, 3. Mu.L 100. Mu.M primer with 9 random nucleotide sequences.
According to an embodiment of the present invention, the second chain extension reaction is performed at 35 to 38℃for 100 to 150 minutes.
In an alternative embodiment of the invention, the nucleotide sequence of the primer with 9 random nucleotide sequences is shown as TCAGACGTGTGCTCTTCCGATCTNNNNNNNNN (SEQ ID NO: 4).
According to an embodiment of the invention, the elution treatment is performed with a potassium hydroxide solution.
In an alternative embodiment of the invention, the concentration of the potassium hydroxide solution is 70 to 90mM.
According to an embodiment of the present invention, the 3 'end to 5' end of the second strand sequence sequentially includes the complementary strand of the adaptor sequence 1, the complementary strand of the sequencing primer 1, the complementary strand of the space coding sequence sequencing primer, the complementary strand of the adaptor sequence 2, the complementary strand of the targeting probe or poly-T probe, the complementary strand of the gene sequence to be bound (or cDNA obtained by reverse transcription of RNA to be bound), and the nucleotide sequence of the primer with 9 random nucleotide sequences.
The scheme of the present invention will be explained below with reference to examples. It will be appreciated by those skilled in the art that the following examples are illustrative of the present invention and should not be construed as limiting the scope of the invention. The examples are not to be construed as limiting the specific techniques or conditions described in the literature in this field or as per the specifications of the product. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.
Example 1:
1. referring to fig. 1, the chip is prepared as follows:
(1) The capture probe seed library hybridized, bridge amplified (see specifically SBS second generation sequencing): on a chip (the chip contains flowcell capable of carrying out flow reaction, the surface of the flowcell is connected with a connector sequence 1 and a connector sequence 2 which are used for hybridization of a seed library), a capture probe seed library is hybridized, bridge amplification is carried out, so that each oligonucleotide cluster grows on the chip, each oligonucleotide cluster contains a plurality of oligonucleotide sequences with consistent sequences, the schematic diagram of the oligonucleotide sequences is shown in figure 2, and the oligonucleotide sequences sequentially comprise the connector sequence 1, a sequencing primer (namely the sequencing primer of figure 2), a space coding sequence sequencing primer and the connector sequence 2 from the 5 'end to the 3' end.
The oligonucleotide sequences were:
CAAGCAGAAGACGGCATACGAGATTCTTTCCCTACACGACGCTCTTCCGATCTNN NNNNNNNNNNNNNNNNNNNNNNNNNNNNCCTCCCACCCTCGCTTGCCCTGCCTCCA ATGGAGATATAATACTCCGGCATTGTGTG(SEQ ID NO:1)。
(2) Space-coding sequencing (see in particular SBS second generation sequencing): primer hybridization, sequencing of the spatial coding sequences, and spatial positioning of each spatial code according to the images to obtain spatial coordinates.
(3) Washing off spatially encoded sequencing chains (see in particular SBS second generation sequencing): spatially encoded sequenced DNA strands are washed off with denaturing reagents and washed with washing reagents.
(4) Ligating the probe sequences; a targeting probe (16S rRNA probe, 5'phos-GACTTTCACCAGTCCATGATCACGGCCCAGACTCCTACGGGAGGCAGCAGT (SEQ ID NO: 6)) of the targeting oligonucleotide sequence (ATCATGGACTGGTGAAAGTCCACACAATGCCGGAGTATTATATCTCCATTG (SEQ ID NO: 5)) and the capture sequence 16S rRNA, a non-targeting probe (poly-T probe, 5' phos-GACTTTCACCAGTCCATGATTTTTT TTTTTTTTTTTTTTTTTTTTTTTTTVN (SEQ ID NO: 7)) of the capture sequence poly-T were added, ligation was performed by T4 ligase reaction (T4 ligase 4U/. Mu.L, 2. Mu.M of the targeting nucleotide sequence, 2. Mu.M of poly-T probe, 0.2. Mu.M of 16S rRNA probe, 1X T4 ligase reaction solution, 37 ℃ overnight reaction). The leader sequence is then washed off with denaturing reagents and three times with washing reagents.
2. Referring to FIG. 3, the steps of capturing, pooling and sequencing mRNA are as follows:
(1) Tissue section, patch: the colon tissue of the mice was cut into 10 μm thick slices on a cryostat and attached to the chip with the targeting probe prepared in step 1 of this example.
(2) Tissue fixation: pre-cooling methanol to-20 ℃, and fixing the chip with the tissue in the step (1) in ice methanol for 30 minutes.
(3) Bacterial cell wall digestion, tissue permeabilization: the tissue on the chip of step (2) was treated with lysozyme (lysozyme, 200. Mu.g/. Mu.L) (room temperature, 1 h), followed by washing with 0.1 Xsodium citrate buffer (SSC); then, treatment (37 ℃ C., 10 minutes) was performed with pepsin (pepsin, 1mg/mL pepsin dissolved in 0.1M HCl and the final concentration of pepsin in solution was 1 mg/mL). Followed by washing with 0.1 XSSC.
(4) Reverse transcription: the chip obtained in step (3) and a reverse transcription mixing reagent (2. Mu.L Maxima H-RTase, 1. Mu.L LRNase inhibitor, 8. Mu.L 5X Maxima 5x RT Buffer,4. Mu.L 10mM dNTP mix, 8. Mu.L 20% Ficoll PM-400,4mL Actinomycin D,13. Mu. L Nuclease free water) were mixed and reacted overnight at 42 ℃. After the completion of the reaction, the reaction solution was discarded, and an exonuclease I solution (Exoclease I, prepared by dissolving exonuclease I in 1 XExoI buffer, the final concentration of exonuclease I was 1U/. Mu.L) was added thereto, followed by a reaction at 37℃for 45 minutes.
(5) Tissue digestion, washing of mRNA chains: the tissue on the chip of step (4) was treated with 80mM KOH, reacted at room temperature for 5 minutes, followed by washing with 0.1 XSSC.
(6) Second strand synthesis complementary to cDNA: the chip obtained in step (5) was mixed with a second strand synthesis mixture (3. Mu.L Klenow fragment, 3. Mu.L NEB buffer, 3. Mu.L 10mM dNTP mix, 3. Mu.L 100. Mu.M primer with 9 random nucleotide sequences (TCAGACGTGTGCTCTTCCGATCTNNNNNNNNN (SEQ ID NO: 4)), 18. Mu.L nucleic-free water), and reacted at 37℃for 2 hours. Washing with ultrapure water 3 times.
(7) Second strand elution: the chip of step (6) was treated with 80mM KOH and reacted at room temperature for 10min, and a KOH solution containing a second strand comprising the complementary strand of linker sequence 1, the complementary strand of sequencing primer 1, the complementary strand of spatially encoded sequence sequencing primer, the complementary strand of linker sequence 2, the complementary strand of targeting probe or poly-T probe, the complementary strand of cDNA to be bound to mRNA obtained by reverse transcription treatment and the nucleotide sequence of the primer having 9 random nucleotide sequences was collected and neutralized with Tris-HCl (1M, pH=7.0).
(8) Library building and sequencing: the second strand product was PCR amplified using Kapa Hifi Hotstart Readymix, the PCR procedure set up to: 95℃for 3 minutes, 15 cycles (95℃for 30 seconds, 60℃for 1 minute, 72℃for 1 minute), 72℃for 2 minutes, 4℃final temperature. The PCR product was purified using 1.2 XAMPure XP magnetic beads.
The product was amplified a second time using Kapa Hifi Hotstart Readymix, and primers were included in the system to add p5, p7 adaptors to each end of the library. The PCR procedure was set as follows: 95℃for 3 minutes, 8 cycles (95℃for 30 seconds, 60℃for 30 seconds, 72℃for 30 seconds), 72℃for 2 minutes, 4℃final temperature. The product was purified using 0.6 XAMPure XP magnetic beads. The library is finally sequenced (space encoding and cDNA, if desired, the cDNA sequencing length can be 50-150 bp).
(9) Data analysis was performed to construct a colon spatial transcription profile and a bacterial spatial distribution profile of the mice, and the results are shown in fig. 4 and 5.
FIG. 4 shows a heat map of mRNA molecule number capture of the small intestine space transcription map, and FIG. 5 shows the abundance distribution of a part of bacteria on the same slice as FIG. 4. Thus, it can be further stated that bacterial mRNA in a tissue sample can be captured using the methods of the present invention, and thus both targeted and non-targeted capture can be achieved.
In the description of the present specification, a description referring to terms "one embodiment," "some embodiments," "examples," "specific examples," or "some examples," etc., means that a particular feature, structure, material, or characteristic described in connection with the embodiment or example is included in at least one embodiment or example of the present invention. In this specification, schematic representations of the above terms are not necessarily directed to the same embodiment or example. Furthermore, the particular features, structures, materials, or characteristics described may be combined in any suitable manner in any one or more embodiments or examples. Furthermore, the different embodiments or examples described in this specification and the features of the different embodiments or examples may be combined and combined by those skilled in the art without contradiction.
While embodiments of the present invention have been shown and described above, it will be understood that the above embodiments are illustrative and not to be construed as limiting the invention, and that variations, modifications, alternatives and variations may be made to the above embodiments by one of ordinary skill in the art within the scope of the invention.

Claims (10)

1. A chip, comprising:
chip substrate, targeting probe and poly-T probe;
wherein the chip substrate comprises a plurality of oligonucleotide sequences, and each of the plurality of oligonucleotide sequences is independently connected with the targeting probe and the poly-T probe;
at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound.
2. The chip of claim 1, wherein the gene sequence to be bound comprises at least one of DNA, lncRNA, mRNA and rRNA;
optionally, the gene sequence to be bound is RNA that does not contain a poly-a tail;
optionally, the ratio of the number of targeting probes to poly-T probes on the chip substrate is (0.1-2): (1-2);
optionally, the ratio of the number of targeting probes to poly-T probes on the chip substrate is (0.1-0.2): (1-2);
optionally, the ratio of the number of targeting probes to poly-T probes on the chip substrate is (0.2-2): (1-2).
3. The chip of claim 1, wherein the targeting probe and/or poly-T probe is linked to the oligonucleotide sequence by a phosphodiester bond;
optionally, the oligonucleotide sequence comprises, in order from the 5 'end to the 3' end, a linker sequence 1, a sequencing primer, a spatial coding sequence sequencing primer, and a linker sequence 2;
optionally, the 5 'end of the targeting probe and/or poly-T probe is linked to the 3' end of the linker sequence 2;
optionally, the 5' end of the linker sequence 1 is attached to the chip substrate.
4. A method of manufacturing a chip, comprising:
ligating the targeting probe and the poly-T probe to a chip substrate comprising a plurality of oligonucleotide sequences to obtain the chip;
wherein at least a portion of the sequence of the targeting probe is complementarily paired with the gene sequence to be bound.
5. The method of claim 4, wherein the gene sequence to be bound comprises at least one of DNA, lncRNA, mRNA and rRNA;
optionally, the gene sequence to be bound is RNA that does not contain a poly-a tail.
6. The method of claim 4 or 5, wherein the oligonucleotide sequence comprises, in order from the 5 'end to the 3' end, a linker sequence 1, a sequencing primer, a spatial coding sequence sequencing primer, and a linker sequence 2;
optionally, the plurality of oligonucleotide sequences are obtained after a PCR amplification process in which the adaptor sequence 1 and/or the adaptor sequence 2 are used as primers and the complementary strand of the oligonucleotide sequences is used as a template;
optionally, after the PCR amplification process and before the ligation process, the oligonucleotide sequences are spatially encoded sequenced in advance to obtain the spatial coordinates of each oligonucleotide sequence of the chip substrate containing the plurality of oligonucleotide sequences.
7. The method of claim 6, wherein the connection process is performed by:
mixing a guide sequence, a polymerase or ligase, the targeting probe and a poly-T probe with the chip substrate comprising a plurality of oligonucleotide sequences;
subjecting the mixed reaction product to denaturation treatment so as to obtain the chip.
8. The method according to claim 7, wherein the mixing reaction is carried out at 35-38 ℃ for 0.5-12 hours;
optionally, the addition amount of the ligase or the polymerase is 3-5U/mu L;
optionally, the molar ratio of the guide sequence, targeting probe and poly-T probe is (1-2): (0.1-2): (1-2);
optionally, the guide sequence comprises a complementary sequence 1 and a linking sequence 1, at least part of the complementary sequence 1 being complementary paired with at least part of the linker sequence 2, at least part of the linking sequence 1 being complementary paired with at least part of the targeting probe and/or poly-T probe;
optionally, the targeting probe and/or poly-T probe comprises a linker sequence 2 and a targeting sequence and/or poly-T sequence, the 3 'end of the linker sequence 2 being linked to the 5' end of the targeting sequence and/or poly-T sequence, at least part of the linker sequence 2 being complementarily paired with at least part of the linker sequence 1.
9. A method of spatial transcriptomics sequencing comprising:
performing spatial transcriptomic sequencing on a tissue sample to be tested using the chip of any one of claims 1-3 or the chip obtained according to the method of any one of claims 4-8;
optionally, the tissue sample to be tested is subjected to a slicing process in advance.
10. The method of claim 9, wherein the spatial transcriptomic sequencing is performed by:
contacting the tissue sample to be tested with a targeting probe and/or a poly-T probe on the chip;
sequentially carrying out tissue fixation treatment and tissue permeabilization treatment on the contact treatment product;
sequentially performing reverse transcription treatment and RNA denaturation removal treatment on the tissue permeabilization treatment product;
sequentially carrying out second chain extension reaction and elution treatment on the RNA denaturation removal treatment product so as to obtain a second chain, wherein the second chain sequence comprises information of a gene sequence to be combined;
carrying out library building and sequencing treatment on the second chain;
and analyzing the data obtained by the library construction and sequencing treatment so as to obtain a spatial transcription map of a sample to be detected or a spatial distribution map of a host of the gene sequence to be combined.
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