CN110724733A

CN110724733A - Sequencing chip for high-resolution space transcriptome

Info

Publication number: CN110724733A
Application number: CN201911047068.4A
Authority: CN
Inventors: 赵祥伟; 叶凯强; 葛芹玉; 汪柳菁
Original assignee: Southeast University
Current assignee: Southeast University
Priority date: 2019-10-30
Filing date: 2019-10-30
Publication date: 2020-01-24
Anticipated expiration: 2039-10-30
Also published as: CN110724733B

Abstract

The spatial transcriptome sequencing technology of the tissue has great significance for biological science and medical research such as cell differentiation, pathogenic mechanism and the like; the patent develops a preparation method of a high-resolution tissue space transcriptome messenger ribonucleic acid sequencing chip based on a micron-sized light cone, the sequencing chip combines a section of mRNA capture probe containing a space position label and reverse transcription oligothymine nucleotide at different optical fiber positions of the end face of the light cone, and the chip constructed by the method ensures that each optical fiber unit in the light cone is provided with a specific position label at the end face to mark the position information of the captured mRNA; the tissue slice is attached to the light cone chip, the transcriptome mRNA in the tissue cell is combined with the capture probe on the light cone chip after permeabilization, then the transcriptome is amplified through technologies such as reverse transcription, in-vitro amplification, polymerase chain reaction and the like, and finally sequencing and analysis are carried out through an Illumina sequencing platform.

Description

Sequencing chip for high-resolution space transcriptome

Technical Field

The invention relates to a sequencing chip for a high-resolution space transcriptome and an application method thereof, belonging to the technical field of biology.

Background

Compared with the traditional cell analysis technology, the single cell analysis realizes the detection of the single cell, highlights the heterogeneity research of the cell, and has important significance for researching the regulation and control mechanism of molecules inside and outside the cell, the cell differentiation process and the cancer cell formation process. The reason for the cellular heterogeneity is that specific transcription in the gene information stream forms a different mRNA system, the cellular transcriptome, that is more spatial and temporal compared to the genome. The mainstream technology for analyzing the single-cell transcriptome at present comprises a single-molecule fluorescence in situ hybridization (smFISH) technology and a single-cell mRNA sequencing technology, and compared with the single-molecule fluorescence in situ hybridization technology, the single-cell mRNA sequencing technology only has the limitation of analyzing a known sequence, and realizes the analysis of the whole transcriptome theoretically. Single cell analysis can be broadly divided into 4 sections of single cell efficient isolation, Whole Genome Amplification (WGA), sequencing and sequence data analysis. At present, parallel large-scale single cell separation is realized based on microfluidics and micropore arrays, and single cell combined labeled sequencing is realized by utilizing bar code mRNA capture beads.

However, single cell sequencing realized by single cell separation loses the original position information of cells in tissues, and the position information of the cells has important significance for researching the microenvironment, the cell differentiation and formation and the regulation and control mechanism among the cells. Therefore, the invention describes a high-resolution space transcriptome sequencing chip based on a light cone and application thereof, so that the transcription information in tissues has space position information and resolution from multi-cell to subcellular level. The invention is beneficial to better research on gene expression and histopathological molecular mechanism of cells in tissues so as to more comprehensively understand the functions of tissues and organs.

Disclosure of Invention

Aiming at the defects existing in the sequencing method and technology of tissue space transcriptomics at present, the invention designs a chip for sequencing a high-resolution space transcriptome based on a light cone, and aims to provide a sequencing method and technology capable of realizing the high-resolution space transcriptome of a tissue slice. By using the method, mRNA in cells in a tissue section can be captured in situ so as to be connected to the surface of a single optical fiber, and then transcriptome sequence information with tissue space position information can be obtained through reverse transcription and sequencing processes.

In order to achieve the purpose, the technical scheme adopted by the invention is as follows:

the chip for sequencing the high-resolution space transcriptome based on the optical fiber light cone is prepared by the following steps:

the chip is a reduced end face of an optical fiber bundle cone (hereinafter referred to as an optical cone), a certain number of optical fibers are tightly arranged into a bundle, the optical fiber bundle can be packaged into a round or polygon from the outer side, the optical fiber bundle is drawn into a cone with a large end and a small end by a melt-drawing method, then the optical fiber bundle is cut in the direction perpendicular to the axis of the optical fiber bundle at the two ends of the cone respectively, and the optical fiber bundle cone with the two ends being a large smooth plane and a small smooth plane can be obtained after the steps of polishing, cleaning and the like, and the reduced side plane is the reduced end face of the optical cone.

The chip of the invention is used for modifying nucleic acid sequences on the reduced end face of the drawn tapered optical fiber. The drawing process comprises the following steps: the method comprises the steps of tightly arranging a certain number of optical fibers into a bundle, packaging the optical fiber bundle into a circle or a polygon from the outer side, placing the optical fiber bundle in a laser drawing instrument, fixing two ends of the optical fiber bundle, setting parameters to start drawing, melting and softening the optical fiber bundle at a laser heating part, applying tension to two ends of the optical fiber bundle, drawing the optical fiber bundle into a cone with one large end and one small end, cutting the optical fiber bundle at two ends of the cone in a direction perpendicular to the axis of the optical fiber bundle, polishing, cleaning and the like to obtain an optical fiber bundle cone with two large and small smooth planes at two ends, namely an optical cone, wherein the plane at one reduced side is the reduced end face of the optical cone. The scheme adopts an in-situ light deprotection group synthesis method, can simultaneously modify the surfaces of different single optical fibers in a light cone with capture probe sequences with different space coding sequences, firstly hydroxylates the drawn reduced end face of the light cone and protects the reduced end face by using a photolysis protecting group, and then places the reduced end face of the light cone in a reaction tank for preparing in-situ synthesis of nucleotide sequences.

A chip for sequencing high-resolution space transcriptome comprises an optical fiber bundle cone, wherein the optical fiber bundle cone is composed of at least 2 optical fibers, two ends of the optical fiber bundle cone are two end faces with a large end face and a small end face, and a nucleotide sequence for coding the position information of captured mRNA is chemically modified on the surface of the optical fiber of the reduced end face of the optical fiber bundle cone.

Further, the protecting group of the reaction site for nucleic acid sequence synthesis may be a plurality of photolabile groups, such as the fiber end face in the reduced end face of the light cone is hydroxylated and protected by a photosensitive protecting group of 4, 4' -Dimethoxytrityl (DMT), or other photosensitive protecting groups attached to the reduced end face of the light cone after different treatments, such as: dithiothreitol (DTT), 2- (2-nitrophenyl) propyl chloroformate (NPPOC), alpha-methyl-6-nitro piperonyloxycarbonyl (MeNPOC), and the like.

Further, the diameter of the optical fibers is 1 μm to 500 μm, and the optical fibers are arranged in a hexagonal shape in the taper.

Furthermore, the nucleotide sequence modified on the end face of the single optical fiber is obtained by an in-situ light deprotection group synthesis method, namely, a light source can be conducted to the surface of the optical fiber at a specific position of the reduced end face in a specific optical fiber through the control of a light cone amplification end face light source, so that the protection group on the position is removed, and the generation of a nucleic acid sequence synthesis reaction on the position is activated.

Furthermore, on the amplifying end face of the light cone, the ultraviolet light source can be projected onto the optical fibers on different specific positions of the amplifying end face simultaneously through controlling the opening and closing of the light holes in the photomask. Thus, the light source at that location will be transmitted through the optical fiber to the corresponding optical fiber in the tapered end face. At this point, the protecting group is removed, and the coupling reaction with the specific base existing in the reaction cell can be carried out, so that the specific base is connected to the surface of the specific optical fiber, and the base also has a photolytically dissociable protecting group, thereby ensuring that only one base is synthesized.

Furthermore, by repeating the steps, the opening and closing of the mask hole and the replacement of the specific base in the reaction tank can be controlled according to a certain program, so that capture sequences with different coding sequence information are synthesized on the surfaces of different single optical fibers in the light cone reduced end face. Since the sequence synthesis on the surface of each fiber is programmed, the sequence information on the individual fibers can be correlated to the position information on the end face of the optical taper after synthesis.

Furthermore, the light cone narrowing end face is a smooth plane, and the micro-pore array formed after chemical corrosion or the micro-pore array containing microspheres is any one of the micro-pore array and the micro-pore array.

Furthermore, a probe sequence is formed on the surface of the optical fiber after the reduced end surface of the optical fiber is subjected to an in-situ optical deprotection group synthesis method, and the sequence structure of the probe sequence comprises a cutting sequence, a sequencing and amplification joint sequence, a spatial position coding sequence, a molecular tag sequence and a capture sequence. The probe sequence is a nucleic acid sequence with a certain length and sequence order formed by in-situ synthesis of different nucleotides on a single optical fiber end face on the optical fiber end face. The nucleic acid sequence of a certain length has different functions in the course of downstream experiments due to different sequence compositions at different positions on the sequence, and thus is given different names. Because the tapered end face is formed by the end faces of the optical fiber bundles, the tapered end face includes the end faces of the optical fibers.

Furthermore, when the chip is used for carrying out tissue slice space transcriptome sequencing, the tissue slice is attached to the reduced end face of the light cone and then is subjected to the steps of permeabilization hybridization, reverse transcription, in-vitro amplification, sequencing and the like to obtain corresponding sequencing data.

Furthermore, after the sequencing data are obtained, the sequencing data with the same spatial position coding sequence are mRNA sequence information captured on the surface of the same optical fiber, and different sequencing information can be positioned to different single optical fiber positions on the end face of the optical cone through data analysis, so that the original position of the optical cone in the tissue is further positioned, and finally tissue spatial transcription map information is obtained.

Compared with the prior art, the invention has the following beneficial effects because the technology is adopted:

the method can conveniently and simultaneously synthesize nucleic acid sequences with different sequence information on each optical fiber end face in the light cone reduced end face and is used for spatial transcriptome sequencing, and the chip can be used for acquiring the transcriptome information in situ in a tissue slice and has the space resolution of a subcellular level, so that the gene expression conditions of cells at different positions in the tissue slice are analyzed, and the method is favorable for researching the heterogeneity and the interrelation among the cells and the molecular mechanism process of the occurrence and the development of diseases.

Description of the drawings:

FIG. 1 is a schematic structural diagram of a light cone structure;

FIG. 2 is a schematic structural diagram of the construction of a photochemical probe modification platform;

FIG. 3 is a diagram showing in-situ deprotection group synthesis for activating a nucleic acid sequence by controlling the opening and closing of the light holes of a mask plate and transmitting a light source to the surface of an optical fiber at a specific position in the reduced end face of a light cone through the optical fiber;

FIG. 4 is a schematic diagram of in situ capture of mRNA in tissue sections by the reduction end of a light cone chip;

FIG. 5 is a flow chart of in vitro amplification from mRNA capture to nucleic acid (different numerical labels indicate different nucleic acid strands); wherein 0 represents a modified primer; 1 represents mRNA captured from a tissue; 2 denotes a reverse transcribed cDNA first strand; 3 represents degraded RNA; 4 represents the second strand of the cDNA; 5 represents the RNA amplified in vitro, and the circled position represents the position of the T7 promoter.

Detailed Description

The invention will be further described with reference to some embodiments, but the claims are not limited to the following examples.

Example 1

Preparation of optical fiber bundle cone

The method comprises the steps of tightly arranging a certain number of optical fibers into a bundle at the reduced end face of an optical fiber bundle cone (hereinafter referred to as an optical cone), packaging the optical fiber bundle into a circular or polygonal shape from the outer side, drawing the optical fiber bundle into a cone with one large end and one small end by a melt drawing method, cutting the optical fiber bundle at the two ends of the cone in the direction perpendicular to the axis of the optical fiber bundle, polishing, cleaning and the like to obtain the optical fiber bundle cone with two large and small smooth planes at the two ends, wherein the plane at one reduced side is the reduced end face of the optical cone.

Example 2

Surface modification of light cone chip

The surface of the light cone chip can be smooth or etched (formed by chemical corrosion), the reduced end face of the light cone after the drawing treatment is subjected to hydroxylation by piranha solution, the end face of the light cone is thoroughly cleaned by ethanol and ultrapure water, and then the hydroxyl is protected by DMT photosensitive protecting groups.

The light cone shrinking surface is arranged in a nucleic acid sequence synthesis reaction pool, and the ultraviolet light source can be simultaneously projected onto optical fibers at different specific positions of the amplifying end surface on the amplifying end surface through controlling the opening and closing of the light holes in the photomask. Thus, the light source at that location will be transmitted through the optical fiber to the corresponding optical fiber in the reduced end face. At this point, the protecting group is removed, and the coupling reaction with the specific base existing in the reaction cell can be carried out, so that the specific base is connected to the surface of the specific optical fiber, and the base also has a photolytically dissociable protecting group, thereby ensuring that only one base is synthesized.

By repeating the steps, the opening and closing of the mask hole and the replacement of the specific base in the reaction tank can be controlled according to a certain program, so that capture sequences with different coding sequence information are synthesized on the surfaces of different single optical fibers in the light cone reduced end face. Since the sequence synthesis on the surface of each fiber is programmed, the sequence information on the individual fibers can be correlated to the position information on the end face of the optical taper after synthesis.

Example 3

Obtaining tissue spatial transcription map information

Taking out the embedded mouse brain tissue from a refrigerator at minus 80 ℃, placing the mouse brain tissue in the refrigerator at minus 20 ℃ for 2 hours, transferring the mouse brain tissue into a freezing slicer, setting slicing parameters, slicing, attaching the sliced tissue slice with the thickness of 10 micrometers to the surface of a light cone chip of an mRNA capture sequence with different space position coding information, and transferring the chip into a clean bench for the next experiment after tissue attachment is completed.

The chip with the tissue section attached is placed in a clean bench for tissue permeabilization, and is treated with type I exonuclease buffer at 37 ℃ for 20 minutes, and then treated with pepsin at 37 ℃ for 15 minutes. After the permeabilization, a reverse transcription step was performed to synthesize a cDNA strand using the captured mRNA as a template, then the tissue section was digested with proteinase K at 56 ℃ for 1 hour, the tissue was cleaned, the cDNA strand was cleaved from the fiber with a cleavage enzyme, and collected in a centrifuge tube.

And performing library construction and on-machine sequencing processes on the collected cDNA sequences, finally positioning the cDNA sequence information connected with each position space coding sequence to a corresponding single optical fiber position on a chip by analyzing the obtained sequence information, after positioning all the information, typing the tissue cells at different positions by software, and visualizing the tissue morphology contour.

Claims

1. A chip for high resolution spatial transcriptome sequencing, characterized in that: the surface of the optical fiber constituting the reduced end face of the optical cone is chemically modified with a nucleotide sequence for encoding positional information of the captured mRNA.

2. The high resolution spatial transcriptome sequencing chip of claim 1, wherein: the modified nucleotide sequence on the surface of a single optical fiber is obtained by an in-situ photo-deprotection group synthesis method.

3. The high resolution spatial transcriptome sequencing chip of claim 2, wherein the in situ photorealistic deprotection group synthesis method comprises the following steps: the light source is transmitted to the light cone reduced end face in the optical fiber under the control of the light cone amplification end face light source, so that the protective group on the light cone reduced end face falls off, and the generation of the nucleic acid sequence synthesis reaction on the surface of the optical fiber is activated.

4. The high resolution spatial transcriptome sequencing chip of claim 3, wherein: the protective group is any one of a photolabile group and a photosensitive protective group.

5. The high resolution spatial transcriptome sequencing chip of claim 4, wherein: the photosensitive protecting group is any one of 4, 4' -dimethoxytrityl, dithiothreitol, 2- (2-nitrophenyl) propyl chloroformate and alpha-methyl-6-nitro piperonyl oxycarbonyl.

6. The high resolution spatial transcriptome sequencing chip of claim 3, wherein: after the light cone reduced end surface is subjected to an in-situ light deprotection group synthesis method, a probe sequence is formed on the surface of the optical fiber, and the sequence structure of the probe sequence comprises a cutting sequence, a sequencing and amplification joint sequence, a spatial position coding sequence, a molecular tag sequence and a capture sequence.

7. The high resolution spatial transcriptome sequencing chip of claim 1, wherein: the light cone reducing end face is a smooth plane, and the micro-pore array or the micro-pore array containing the microspheres is formed after chemical corrosion.

8. The high resolution spatial transcriptome sequencing chip of claim 1, wherein: the diameter of each optical fiber is 1-500 μm, and the optical fibers in the light cone are arranged in a hexagonal shape.

9. Use of the high resolution spatial transcriptome sequencing chip of claims 1-8 for obtaining tissue spatial transcription profile information.

10. The use of claim 9, wherein the step of obtaining tissue spatial transcription profile information is as follows:

(1) when the chip is used, the capture probe fixed on the surface of each optical fiber on the end face of the light cone and mRNA in the tissue are subjected to in-situ hybridization and combination, and transcriptome mRNA in the tissue is fixed at the end face position of the optical fiber;

(2) synthesizing complementary sequence cDNA from the captured mRNA in a reverse transcription reaction system, recovering nucleic acid, and performing high-throughput sequencing after in vitro transcription and PCR amplification;

(3) after the sequencing data are obtained, the sequencing data with the same spatial position coding sequence position the sequencing information to the position of a single optical fiber on the end face of the optical cone through data analysis, so that the original position of the single optical fiber in the tissue is positioned, and finally, the tissue spatial transcription map information is obtained.