Spatial transcriptome micro-fluidic chip with high resolution and high throughput position information and preparation method of spatial transcriptome micro-fluidic chip

Abstract

The invention discloses a spatial transcriptome micro-fluidic chip with high-resolution and high-throughput position information and a preparation method thereof. The space transcriptome micro-fluidic chip comprises a micro-pit array layer, a space bar code X coding layer, a space bar code Y coding layer and a space bar code Z coding layer, and by means of the space transcriptome micro-fluidic chip, 100 kinds of coding sequences on the three dimensions of X, Y and Z can be sequentially connected to form 1 million kinds of space bar code information through simple operation steps. The method is used for recording position information corresponding to one million micro pits, the resolution ratio reaches 10 micrometers or below, the dot matrix number reaches million level, and the method has good application prospects in space transcriptome sequencing based on space bar codes.

Description

Technical Field

[0001] The present invention relates to the field of microfluidic technology, and in particular to a spatial transcriptome microfluidic chip with high-resolution and high-throughput position information and a preparation method thereof. Background Art

[0002] Spatial transcriptome sequencing technology can simultaneously obtain the spatial location information and gene expression data of cells, which has promoted the study of the true gene expression of cells in situ in tissues, and provided an important research method for many fields such as tissue cell function, microenvironment interaction, lineage tracing of developmental processes, and disease pathology. At present, there are two main methods of spatial transcriptome, one is based on spatial barcodes, and the other is based on in situ transcriptome sequencing. Among them, spatial transcriptome sequencing based on spatial barcodes is mainly based on mRNA capture array chips, which are modified with thousands of capture probe cluster arrays containing spatial location information. After the tissue sections are permeabilized, the intracellular mRNA is captured by the oligo-dT sequence at the end of the spatial transcriptome chip probe, and then cDNA is synthesized using mRNA as a template. After the library is constructed, it is sequenced to obtain the location information of gene expression. However, the number of dots and resolution (size) of existing spatial transcriptome chips are important factors in the difficulty and cost of their preparation. In actual applications, chip structures with a large number of dots and high resolution are conducive to obtaining more and more accurate biological information. However, there is still a lack of spatial transcriptome chips with a resolution of 10μm or less and a dot number of millions on the market. Summary of the invention

[0003] The purpose of the present invention is to provide a spatial transcriptome microfluidic chip with high resolution and high throughput position information and a preparation method thereof in view of the shortcomings of the spatial transcriptome chips currently on the market.

[0004] To achieve the above object, the present invention provides the following technical solutions: In a first aspect, a device for preparing a spatial transcriptome microfluidic chip is provided, comprising the following structural layers:

[0005] A micropit array layer, comprising N large arrays located in the middle region, each large array comprising N rows and N columns of micropits;

[0006] The spatial barcode X coding layer comprises N inlets and N outlets located on both sides, and N vertical microchannels located in the middle area and connecting the inlets and outlets; when the spatial barcode X coding layer is aligned with the micropit array layer, each vertical microchannel flows through all large arrays, flows through N micropits in each large array, and each micropit has only one vertical microchannel flowing through it;

[0007] The spatial barcode Y coding layer comprises N inlets and N outlets located on both sides, and N transverse microchannels located in the middle area and connecting the inlets and outlets; when the spatial barcode Y coding layer is aligned with the micropit array layer, each transverse microchannel flows through all the large arrays, flows through N micropits in each large array, and each micropit has only one transverse microchannel flowing through it;

[0008] The spatial barcode Z coding layer comprises an isolation fence located in the middle area; when the spatial barcode Z coding layer is aligned with the micro-pit array layer, the isolation fence is used to separate the N large arrays;

[0009] Wherein, N is a natural number.

[0010] Preferably, N=100.

[0011] Furthermore, the micro-pits are in the shape of cylinders or cubes.

[0012] Furthermore, when the micro-pit is cylindrical, the width of the transverse micro-channel or the vertical micro-channel is equal to the diameter of the micro-pit; when the micro-pit is a cube, the width of the transverse micro-channel or the vertical micro-channel is equal to the length and width of the micro-pit. This design allows the micro-channel to be aligned with the micro-pit it flows through.

[0013] Preferably, the material of the micropit array layer is PDMS, and the material of the spatial barcode X coding layer, the spatial barcode Y coding layer, and the spatial barcode Z coding layer is COC, PMMA, PC or glass.

[0014] In a second aspect, a method for preparing a spatial transcriptome microfluidic chip based on the device is provided, comprising the following steps:

[0015] Place magnetic beads in each micro-pit of the micro-pit array layer, align the spatial barcode X-coding layer with the micro-pit array layer, add N different X-coding DNA sequences to the N inlets of the spatial barcode X-coding layer, and fill all micro-pits and vertical micro-channels so that the X-coding DNA sequence is connected to the magnetic beads, and wash;

[0016] Replace the spatial barcode Y coding layer and align it with the micro-pit array layer, add N different Y coding DNA sequences to the N inlets of the spatial barcode Y coding layer, and fill all the micro-pits and lateral micro-channels, so that the Y coding DNA sequence is connected to the X coding DNA sequence on the magnetic beads, and wash;

[0017] The spatial barcode Z coding layer is replaced and aligned with the micropit array layer, and N different Z coding DNA sequences are added to the N large arrays separated by the spatial barcode Z coding layer, and all the micropits are filled, so that the Z coding DNA sequence is connected to the Y coding DNA sequence on the magnetic bead; 3Magnetic bead micropit array with position information for spatial transcriptome sequencing;

[0018] The space is divided into X, Y, and Z plane dimensions; the X-encoding DNA sequence, the Y-encoding DNA sequence, and the Z-encoding DNA sequence are used to record position information in the X, Y, and Z plane dimensions, respectively.

[0019] Furthermore, the magnetic beads and the X-coding DNA sequence are both modified with chemical groups, and the magnetic beads are connected to the X-coding DNA sequence through chemical group coupling;

[0020] When the Y-coding DNA sequence is added, the method further includes: adding a PCR amplification system so that the Y-coding DNA sequence is connected to the X-coding DNA sequence on the magnetic beads after the reaction;

[0021] When the Z-coding DNA sequence is added, the method further comprises: adding a PCR amplification system so that the Z-coding DNA sequence is connected to the Y-coding DNA sequence on the magnetic beads after the reaction.

[0022] In a third aspect, a spatial transcriptome microfluidic chip with high-resolution and high-throughput positional information prepared by the preparation method is provided.

[0023] The beneficial effects of the present invention are as follows:

[0024] The present invention prepares a new spatial transcriptome microfluidic chip, which can connect 100 coding sequences in three dimensions of X, Y, and Z in sequence to form 1 million spatial barcode information by simple operation steps, and is used to record the position information corresponding to 1 million micropits. The resolution reaches 10 μm or less, and the number of dot arrays reaches the million level. It has good application prospects in spatial transcriptome sequencing based on spatial barcodes. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 is a schematic diagram of the micro-pit array layer;

[0026] Figure 2 Schematic diagram of the spatial barcode X coding layer; (a) is the complete structure; (b) is a schematic diagram of a flow path;

[0027] Figure 3 Schematic diagram of the Y coding layer of the spatial barcode; (a) is the complete structure; (b) is a schematic diagram of a flow path;

[0028] Figure 4 is a schematic diagram of the Z coding layer of the spatial barcode;

[0029] Figure 5 It is a schematic diagram of the spatial barcode synthesis process. DETAILED DESCRIPTION

[0030] The present invention develops a spatial transcriptome microfluidic chip with high resolution and high throughput position information and a preparation method thereof, comprising four structural layers: a micropit array layer, a spatial barcode X coding layer, a spatial barcode Y coding layer, and a spatial barcode Z coding layer.

[0031] The micropit array layer includes 10×10 large arrays, each large array includes 100×100 micropit arrays, and the micropits are circular micropits with a diameter of 10 μm and a depth of 10 μm or square micropits with a length, width and depth of 10 μm. The center distance of the micropits is 19 μm. Figure 1 As shown, it contains 1 million micro-pits. The spatial barcode X coding layer includes 100 inlets, 100 outlets, and 100 vertical micro-channels connecting the inlets and outlets ( Figure 2 a), the pipeline is 10μm wide and 10μm deep, with a spacing of 9μm. Each pipeline is used to transport a coding sequence and flows through the array 10 times. The pipeline flow path diagram is shown in Figure 2 b. 100 channels can cover the entire micro-pit array. Similarly, the spatial barcode Y coding layer is a horizontal micro-channel ( Figure 3 The spatial barcode Z encoding layer is a 10×10 large array isolation fence ( Figure 4 ), used to label 100 Z coding sequences. In summary, 100 coding sequences of X, Y, and Z are connected in sequence ( Figure 5 ) constitutes 1 million kinds of spatial barcode information to record the location information corresponding to 1 million micro-pits.

[0032] The preparation process of the spatial transcriptome microfluidic chip is as follows: first, the magnetic beads modified with chemical groups (such as carboxyl) are distributed in the micropits of the micropit array layer. The spatial barcode X coding layer is aligned with the micropit array layer through an alignment device. 100 coding DNA sequences with chemical modifications (such as amino groups) are added to the inlet of the X coding layer. After negative pressure is applied at the outlet to allow the liquid to fill the pipeline and the micropit array, the DNA sequence on the magnetic beads is extended by chemical group coupling to obtain the X coding information. After cleaning, the spatial barcode Y coding layer is replaced, and 100 coding DNA sequences (including PCR amplification system) are added to the inlet. After sealing the chip with a sealing film, several rounds of PCR are performed to extend the DNA sequence on the magnetic beads to obtain the Y coding information. Similarly, the spatial barcode Z coding layer and 100 DNA coding sequences are replaced by PCR to complete the extension of the Z coding information. In summary, the preparation of the magnetic bead micropit array containing 1 million position information is completed for subsequent spatial transcriptome sequencing applications.

[0033] Example 1

[0034] Prepare the PDMS micropit array layer and the spatial barcode X, Y, and Z coding layers of hard materials such as COC and glass. Soak the PDMS micropit array layer in 1% SDS aqueous solution for 5 minutes and blow dry with nitrogen; add 1mg / mL carboxyl magnetic beads activated by EDC / NHS with a particle size of 8μm, and place a magnet on the chip to assist the magnetic beads to settle into the micropits. Align the spatial barcode X coding layer to the micropit array, add the amino-modified X coding sequence at the inlet, and the pressure at the outlet is -600mbar so that the liquid covers the entire micropit array, and incubate at room temperature for 1 hour. Cover the inlet with pure water, continue to give negative pressure at the outlet, and wash for 5 minutes. Uncover the spatial barcode X coding layer on the magnet, align the spatial barcode Y coding layer to the micropit array, add the Y coding DNA sequence containing the PCR amplification system at the inlet, and the pressure at the outlet is -600mbar so that the liquid covers the entire micropit array. The inlet and outlet are sealed with sealing film, placed on a flat PCR, pressed with an insulating block, and run the program in Table 1. Place the chip on a 95°C heating plate, cover the inlet with enzyme-free water, keep negative pressure on the outlet, and wash for 5 minutes. Uncover the spatial barcode Y coding layer on the magnet, align the spatial barcode Z coding layer and stick it on the micro-pit array, add the Z coding DNA sequence in each fence, stick the sealing film, press it with an insulating block on the flat PCR instrument, and run the program in Table 2. Place the chip on a 95°C heating plate, cover the inlet with enzyme-free water, keep negative pressure on the outlet, and wash for 5 minutes. Uncover the spatial barcode Z coding layer on the magnet, which can then be used for the spatial transcriptome sequencing process of the tissue sample.

[0035] Table 1 PCR program 1

[0036]

[0037] Table 2 PCR program 2

[0038] Heating cover temperature Reaction volume Run time 102℃ 10ul ～ step temperature time 1 94℃ 5min 2 48.8℃ 20min 3 72℃ 20min 4 4℃ Hold

[0039] It should be noted that the above is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Without making creative work, any modification, equivalent replacement, improvement, etc., shall be included in the protection scope of the present invention.

Claims

1. A device for preparing a spatial transcriptome microfluidic chip, characterized in that: The following structural layers are included: A micropit array layer, comprising N large arrays located in the middle region, each large array comprising N rows and N columns of micropits; The spatial barcode X coding layer comprises N inlets and N outlets located on both sides, and N vertical microchannels located in the middle area and connecting the inlets and outlets; when the spatial barcode X coding layer is aligned with the micropit array layer, each vertical microchannel flows through all large arrays, flows through N micropits in each large array, and each micropit has only one vertical microchannel flowing through it; The spatial barcode Y coding layer comprises N inlets and N outlets located on both sides, and N transverse microchannels located in the middle area and connecting the inlets and outlets; when the spatial barcode Y coding layer is aligned with the micropit array layer, each transverse microchannel flows through all the large arrays, flows through N micropits in each large array, and each micropit has only one transverse microchannel flowing through it; The spatial barcode Z-coding layer includes the isolation fence located in the middle area; When the spatial barcode Z coding layer is aligned with the micropit array layer, the isolation fence is used to separate the N large arrays; Wherein, N is a natural number.

2. The device according to claim 1, characterized in that The shape of the micro-pit is cylinder or cube.

3. The device according to claim 2, characterized in that When the micropit is cylindrical in shape, the width of the transverse microchannel or the vertical microchannel is equal to the diameter of the micropit; when the micropit is cube-shaped, the width of the transverse microchannel or the vertical microchannel is equal to the length and width of the micropit.

4. The device according to claim 1, characterized in that The material of the micro-pit array layer is PDMS, and the materials of the spatial barcode X coding layer, the spatial barcode Y coding layer, and the spatial barcode Z coding layer are COC, PMMA, PC or glass.

5. A method for preparing a spatial transcriptome microfluidic chip based on the device according to claim 1, characterized in that: The following steps are involved: Place magnetic beads in each micro-pit of the micro-pit array layer, align the spatial barcode X-coding layer with the micro-pit array layer, add N different X-coding DNA sequences to the N inlets of the spatial barcode X-coding layer, and fill all micro-pits and vertical micro-channels so that the X-coding DNA sequence is connected to the magnetic beads, and wash; Replace the spatial barcode Y coding layer and align it with the micro-pit array layer, add N different Y coding DNA sequences to the N inlets of the spatial barcode Y coding layer, and fill all the micro-pits and lateral micro-channels, so that the Y coding DNA sequence is connected to the X coding DNA sequence on the magnetic beads, and wash; The spatial barcode Z coding layer is replaced and aligned with the micropit array layer, and N different Z coding DNA sequences are added to the N large arrays separated by the spatial barcode Z coding layer, and all the micropits are filled, so that the Z coding DNA sequence is connected to the Y coding DNA sequence on the magnetic bead; 3 Magnetic bead micropit array with position information for spatial transcriptome sequencing; The space is divided into X, Y, and Z plane dimensions; the X-encoding DNA sequence, the Y-encoding DNA sequence, and the Z-encoding DNA sequence are used to record position information in the X, Y, and Z plane dimensions, respectively.

6. The preparation method according to claim 5, characterized in that: The magnetic beads and the X-coding DNA sequence are both modified with chemical groups, and the magnetic beads are connected to the X-coding DNA sequence through chemical group coupling; When the Y-coding DNA sequence is added, the method further includes: adding a PCR amplification system so that the Y-coding DNA sequence is connected to the X-coding DNA sequence on the magnetic beads after the reaction; When the Z-coding DNA sequence is added, the method further includes: adding a PCR amplification system so that the Z-coding DNA sequence is connected to the Y-coding DNA sequence on the magnetic beads after the reaction.

7. A spatial transcriptome microfluidic chip with high resolution and high throughput positional information prepared by the preparation method of claim 5.

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