CN114085897A - Space omics sequencing method - Google Patents

Space omics sequencing method Download PDF

Info

Publication number
CN114085897A
CN114085897A CN202111372033.5A CN202111372033A CN114085897A CN 114085897 A CN114085897 A CN 114085897A CN 202111372033 A CN202111372033 A CN 202111372033A CN 114085897 A CN114085897 A CN 114085897A
Authority
CN
China
Prior art keywords
sample
barcode
reaction
sequencing
microfluidic
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202111372033.5A
Other languages
Chinese (zh)
Inventor
彭广敦
江伏青
瞿芳芳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Bioisland Laboratory
Original Assignee
Bioisland Laboratory
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Bioisland Laboratory filed Critical Bioisland Laboratory
Priority to CN202111372033.5A priority Critical patent/CN114085897A/en
Publication of CN114085897A publication Critical patent/CN114085897A/en
Pending legal-status Critical Current

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6804—Nucleic acid analysis using immunogens

Abstract

The embodiment of the disclosure discloses a method for sequencing a space group, which comprises the following steps: pretreating a sample slice; covering the microfluidic chip on the sample slice; adding first substances containing different barcode A into sample inlet holes of the microfluidic chip respectively, and carrying out a first reaction with the sample slices; washing the sample slice with a buffer; rotating the microfluidic chip at a preset angle, so that the direction of a microfluidic channel of the microfluidic chip through which the first substance flows and the direction of the rotated microfluidic channel form the cross positioning mark; adding second substances containing different barcode B into the sample inlet holes of the rotated microfluidic chip respectively, and carrying out a second reaction with the sample slices; wherein the first and second substances further comprise Tn 5; and sequentially carrying out cell lysis, amplification and library establishment on the sample slices, and then carrying out gene sequencing according to the cross-localization markers.

Description

Space omics sequencing method
The application is a divisional application of Chinese patent application 202011057632.3, the application date of the original application 202011057632.3 is 9/29/2020, and the invention is named as a method for space omics sequencing, single cell epigenomics sequencing and location identification.
Technical Field
The disclosure relates to the technical field of gene sequencing, in particular to a space omics sequencing method.
Background
Spatial heterogeneity is a key feature of organ function, and positional information of cells is important for the study of cell regulatory mechanisms and cell lineage development processes. Conventional genetic sequencing is performed on the whole tissue sample or cell population, the differences between cells may be masked by averaging, and the single cell testing technology can reveal genetic information of each cell at the single cell level, so that different cell types can be distinguished. But still lack the ability to reveal information about the original location of the cell within the tissue. Therefore, a sequencing method that retains the original spatial location information of the cells is needed.
Disclosure of Invention
To solve the problems in the related art, embodiments of the present disclosure provide a method of spatial omics sequencing.
The embodiment of the disclosure provides a space omics sequencing method.
Specifically, the space omics sequencing method comprises the following steps:
pretreating a sample slice;
covering the microfluidic chip on the sample slice;
adding first substances containing different barcode A into sample inlet holes of the microfluidic chip respectively, and carrying out a first reaction with the sample slices;
washing the sample slice with a buffer;
rotating the microfluidic chip at a preset angle, so that the direction of a microfluidic channel of the microfluidic chip through which the first substance flows and the direction of the rotated microfluidic channel form the cross positioning mark;
adding second substances containing different barcode B into the sample inlet holes of the rotated microfluidic chip respectively, and carrying out a second reaction with the sample slices; wherein the first and second substances further comprise Tn5, and the Tn5 breaks the chromatin in the partner binding region to obtain a target DNA fragment, and barcode A and barcode B are added to the target DNA fragment;
and sequentially carrying out cell lysis, amplification and library establishment on the sample slices, and then carrying out gene sequencing according to the cross-localization markers.
Optionally, prior to the first reaction, adding a partner for the protein of interest to bind to the protein of interest; optionally, a step of reverse treatment of RNA with primers to obtain cDNA is also included before adding the partner.
Optionally, the method further comprises:
the DNA of the sample sections is digested with a digestion reagent before RNA inversion treatment.
Optionally, pre-treating the sample section comprises at least one of the following:
preparing a sample slice, staining the sample slice, fixing the sample slice, blocking BSA (bovine serum albumin) of the sample slice, performing permeation treatment on the sample slice, and placing the sample slice in a sample area in the middle of a glass slide.
Optionally, the number of the sample outlet holes of the microfluidic chip is less than that of the sample inlet holes, so as to increase the number of the sample inlet holes and the microfluidic channels forming the cross positioning marks.
Optionally, in the process of performing the first reaction and/or the second reaction, the first substance and/or the second substance is/are smoothly caused to enter the sample labeling area by a mode of inflating the sample inlet hole of the microfluidic chip by positive pressure and/or sucking the sample outlet hole of the microfluidic chip by negative pressure; wherein the sample marking area is an area where the microfluidic chip covers the sample slice, and the area is provided with the microfluidic channels which are arranged in parallel; and/or in the process of carrying out the first reaction and/or the second reaction, a hard object penetrates through the anti-backflow hole near the sample outlet hole of the chip to extrude the microfluidic channel, so that the microfluidic channel deforms and completely blocks the channel; and after the reaction is finished, taking out the hard object, and recovering the extruded microfluidic channel by self.
The embodiment of the disclosure also provides a space omics sequencing method.
Specifically, the method for sequencing the space group comprises the following steps:
pretreating a sample slice;
adding a partner of the target protein to the sample section;
covering the microfluidic chip on the sample slice, adding a mixed solution of pre-embedded Tn5-barcode A into the sample inlet, pumping out the sample inlet, and carrying out a first reaction after the microfluidic channel is filled with liquid; said mixed solution of Tn5-barcode a further comprising a partner binding substance for binding a partner of said target protein;
removing the microfluidic chip, washing the sample slice, rotating the microfluidic chip by 90 degrees, covering the microfluidic chip on the sample slice again, adding the mixed solution of pre-embedded Tn5-barcode B into the sample inlet, pumping out the sample inlet, and carrying out a second reaction after the microfluidic channel is filled with liquid; said mixed solution of Tn5-barcode B further comprises a partner binding substance for binding a partner of said target protein;
breaking the chromatin of the sample slice with Tn5 to form DNA fragments while adding barcode A and barcode B on the DNA fragments;
adding EDTA or EGTA into the sample inlet, pumping out the sample inlet until the microfluidic channel is filled with liquid, and reacting at corresponding temperature for a proper time to terminate the reaction;
and dissociating the sample slice by using the lysis solution for subsequent cDNA purification and amplification and library construction sequencing.
Optionally, before adding the partner, a step of obtaining cDNA by reverse processing of RNA by using a primer is also included.
Optionally, the method further comprises:
the DNA of the sample sections is digested with a digestion reagent before RNA inversion treatment.
Optionally, the partner is selected from an antibody, an antibody Fc fragment; the partner binding substance is selected from protein A, protein G, Fc receptor protein.
Optionally, pre-treating the sample section comprises at least one of the following:
the method comprises the following steps of sample slice manufacturing, sample slice staining, sample slice fixing, BSA sealing of the sample slice, sample slice permeation treatment and sample slice placing in a sample area in the middle of a glass slide.
Optionally, the number of the sample outlet holes of the microfluidic chip is less than that of the sample inlet holes, so as to increase the number of the sample inlet holes and the microfluidic channels forming the cross positioning marks.
Optionally, in the process of performing the first reaction and/or the second reaction, the first substance and/or the second substance is/are smoothly caused to enter the sample labeling area by a mode of inflating the sample inlet hole of the microfluidic chip by positive pressure and/or sucking the sample outlet hole of the microfluidic chip by negative pressure; wherein the sample marking area is an area where the microfluidic chip covers the sample slice, and the area is provided with the microfluidic channels which are arranged in parallel; and/or in the process of carrying out the first reaction and/or the second reaction, a hard object penetrates through the anti-backflow hole near the sample outlet hole of the chip to extrude the microfluidic channel, so that the microfluidic channel deforms and completely blocks the channel; and after the reaction is finished, taking out the hard object, and recovering the extruded microfluidic channel by self.
The technical scheme provided by the embodiment of the disclosure can have the following beneficial effects:
the space omics sequencing method of the embodiments of the present disclosure preprocesses a sample slice; covering the microfluidic chip on the sample slice; adding first substances containing different barcode A into sample inlet holes of the microfluidic chip respectively, and carrying out a first reaction with the sample slices; washing the sample slice with a buffer; rotating the microfluidic chip at a preset angle, so that the direction of a microfluidic channel of the microfluidic chip through which the first substance flows and the direction of the rotated microfluidic channel form the cross positioning mark; adding second substances containing different barcode B into the sample inlet holes of the rotated microfluidic chip respectively, and carrying out a second reaction with the sample slices; wherein the first and second substances further comprise Tn5, and the Tn5 breaks the chromatin in the partner binding region to obtain a target DNA fragment, and barcode A and barcode B are added to the target DNA fragment; and sequentially carrying out cell lysis, amplification and library establishment on the sample slices, and then carrying out gene sequencing according to the cross-localization markers. The sequencing method utilizes the microfluidic chip to add substances including different barcode at the marked cell position for space omics sequencing, and has simple operation steps. The micro-fluidic chip used in the testing method has the advantages that the number of the sample outlet holes is less than that of the sample inlet holes, the design that the sample inlet holes are communicated with the sample outlet holes in the past is changed, more than two micro-fluidic channels respectively communicated with the sample inlet holes are communicated with the same sample outlet hole, the number of the sample inlet holes and the micro-fluidic channels is increased, the number of the micro-fluidic channels capable of forming cross positioning marks is increased, the space and the width of the micro-fluidic channels can be further reduced, and therefore the detection flux of the space omics sequencing method and the mark area of a sample are improved.
It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive of the disclosure.
Drawings
Other features, objects, and advantages of the present disclosure will become more apparent from the following detailed description of non-limiting embodiments when taken in conjunction with the accompanying drawings. In the drawings:
fig. 1 shows a schematic structural diagram of a microfluidic chip according to an embodiment of the present disclosure;
fig. 2a shows a schematic structural diagram of a first chip according to an embodiment of the present disclosure;
fig. 2b shows a schematic structural diagram of a second chip according to an embodiment of the present disclosure;
figure 3 shows a schematic flow diagram of a method of spatial omics sequencing according to an embodiment of the present disclosure;
FIG. 4 is a diagram of the structure of the coding sequences for cross-localization markers in sequencing of the spatial transcriptome;
FIG. 5 is a diagram of the structure of the coding sequences for cross-localization markers in sequencing of the spatial proteome;
FIG. 6 is a diagram of the structure of the coding sequences for cross-localization markers in sequencing of the spatial appearance group;
FIG. 7 is a diagram of the structure of the coding sequences for cross-localization markers in sequencing of a spatial epiglotte;
FIG. 8 shows a schematic flow diagram of a method of location identification on a slide according to an embodiment of the present disclosure;
figure 9 shows a schematic flow diagram of a single cell epigenetics sequencing method according to an embodiment of the disclosure.
Detailed Description
Hereinafter, exemplary embodiments of the present disclosure will be described in detail with reference to the accompanying drawings so that those skilled in the art can easily implement them. Also, for the sake of clarity, parts not relevant to the description of the exemplary embodiments are omitted in the drawings.
In the present disclosure, it is to be understood that terms such as "including" or "having," etc., are intended to indicate the presence of the disclosed features, numbers, steps, behaviors, components, parts, or combinations thereof, and are not intended to preclude the possibility that one or more other features, numbers, steps, behaviors, components, parts, or combinations thereof may be present or added.
It should be further noted that the embodiments and features of the embodiments in the present disclosure may be combined with each other without conflict. The present disclosure will be described in detail below with reference to the accompanying drawings in conjunction with embodiments.
The term partner refers to a molecule that binds to a protein of interest, including antibodies, aptamers, antigen-binding fragments, including whole antibodies, recombinant antibodies, or antibody fragments, such as antibody Fc fragments, Fab fragments, scFv, and any of IgM, IgG, IgA, IgD, IgE.
Example one
The space omics sequencing method provided by the embodiment of the disclosure adopts a microfluidic chip technology, and fig. 1 shows a schematic structural diagram of a microfluidic chip according to the embodiment of the disclosure. Fig. 2a shows a schematic structural diagram of a first chip according to an embodiment of the present disclosure. Fig. 2b shows a schematic structural diagram of a second chip according to an embodiment of the present disclosure. As shown in fig. 1, fig. 2a, and fig. 2b, the microfluidic chip 10 includes: a first chip 11, a second chip 12 and a third chip 13. The first chip 11 includes: a sample inlet hole 111, a sample outlet hole 112, and a microfluidic channel 113 communicating the sample inlet hole 111 and the sample outlet hole 112. The number of the sample outlet holes 112 is smaller than that of the sample inlet holes 111, at least more than two microfluidic channels 113 respectively communicated with the sample inlet holes 111 are communicated with the same sample outlet hole 112, and the number of the sample inlet holes 111 and the number of the microfluidic channels 113 are increased on a chip with the same area.
The first chip 11 is provided with a sample labeling area a having microfluidic channels 113 arranged in parallel. The spatial sequencing method provided by the embodiment of the disclosure can be independently completed by using the first chip 11, when in use, the sample marking region a of the first chip 11 covers the sample slice, the microfluidic channel 113 is communicated with the sample slice, and after substances including different barcode (bar codes) are added to the sample inlet 111, the substances can smoothly flow to the sample marking region by pumping out the sample hole 112 to react with the sample slice in the region, so that the barcode is added to the cell material of the sample slice.
The second chip 12 may be bonded to the first chip 11 to be used as a single chip, or may be provided separately, and when in use, the second chip 12 is stacked on the first chip 11 to be used. The material of first chip 11 is polydimethylsiloxane usually, this material is softer easily by rifle head absorption when the application of sample, second chip 12 can adopt the harder material of first chip 11 relatively, for example polymethyl methacrylate, the two cooperation is used, second chip 12 plays the guard action, rifle head and first chip 11 direct contact when avoiding the application of sample, thereby can effectively avoid leading to first chip 11 and the slide glass at sample section place to break away from because of rifle head absorption, prevent that the independent reagent that first chip 11 different sample inlet hole 111 added from taking place to mix and arouse the experiment failure, can also cooperate with row rifle or automatic liquid-transfering workstation, realize the automation of application of sample.
Specifically, the second chip 12 is provided with a first through hole 121, and the position of the first through hole 121 corresponds to the positions of the sample inlet hole 111 and the sample outlet hole 112 on the first chip 11. The second chip 12 is further provided with an anti-reflux hole 122, the anti-reflux hole 122 is located close to the sample outlet 112, and in the process of adding barcode, a hard object can penetrate through the anti-reflux hole 122 to press a region b close to the sample outlet on the first chip 11, so that the microfluidic channel 113 in the region b is deformed to completely block the channel, and the substance is prevented from flowing back from the sample outlet 112. After the reaction is completed, the hard object is removed and the extruded microfluidic channel 113 is self-restored.
The third chip 13 is used in cooperation with the first chip 11, and considering that after the number of the sample injection holes 111 and the number of the microfluidic channels 113 are increased on the first chip 11, the distance between adjacent microfluidic channels 113 in the sample marking region and the width of the microfluidic channels 113 can be set smaller, so that the detection throughput of the sample slice is improved, and the microfluidic channels 113 may be blocked. In order to avoid the substances added into the sampling holes 111 from blocking the microfluidic channel, the third chip 13 is added and superposed on the first chip 11 or the second chip 12 for use, and the third chip 13 promotes the substances added into the sampling holes 111 to smoothly flow to the sample marking area in a manner of inflating the sampling holes 111 with positive pressure. Specifically, the third chip 13 is provided with a second through hole, and the position of the second through hole corresponds to the positions of the sampling hole 112 on the first chip 11 and the anti-backflow hole 122 on the second chip 12. The third chip 13 is further provided with a hollow area corresponding to the position of the sample inlet 111, the hollow area is superposed on the first chip 11 or the second chip 12 to form a closed space, and the hollow area is provided with an inflation hole for inflating the closed space, so as to form positive pressure above the sample inlet 111.
According to embodiments of the present disclosure, the microfluidic channels have a pitch of 100nm-200 μm and/or a width of 100nm-200 μm.
For example, the distance between the microfluidic channels is 10 μm, the width of the microfluidic channels is 20 μm, and it can be realized that 384 sample wells 113 are arranged on a chip area of 11cm × 11cm, so that 147456 label combinations of 384 × 384 are realized, the effective labeling rate for the sample slice is increased to about 44.4%, and the coverage area of the sample slice also reaches 11.5mm × 11.5 mm.
In the present disclosure, the number of the sample injection holes is 10 to 40000. For example, 768 wells are used to achieve 589824 combinations of 768x768, to achieve coverage of a 23.04mm x 23.04mm sample area with a microfluidic channel width of 20 μm and a microfluidic channel width pitch of 10 μm.
Figure 3 shows a schematic flow diagram of a method of spatial omics sequencing according to an embodiment of the present disclosure. As shown in fig. 3, the spatial omics sequencing method comprises steps S110-S130.
In step S110, the sample slice is pretreated;
in step S120, adding a cross-localization identifier to the sample slice using a microfluidic chip, wherein the cross-localization identifier is determined by substances including different barcodes added to a sample inlet of the microfluidic chip;
in step S130, after cell lysis, amplification and library establishment are sequentially performed on the sample slice, gene sequencing is performed according to the cross-localization markers.
According to the space omics sequencing method provided by the embodiment of the disclosure, after a sample slice is pretreated, substances including different barcodes are added from a sample inlet of a microfluidic chip to form a cross location marker for marking a space position of a cell, then the cross location marker is added to cell substances (such as RNA, DNA or protein) of the sample slice, cell lysis, amplification and library building are sequentially carried out, and finally sequencing and data analysis are carried out. The sequencing method utilizes the microfluidic chip to add substances including different barcodes for marking the positions of cells, has simple operation steps, is not limited to space transcriptome sequencing, space proteome sequencing, space epigenome sequencing and the like, and has wide application range. The number of the sample outlet holes of the micro-fluidic chip used in the testing method is less than that of the sample inlet holes, the design that the sample inlet holes are communicated with the sample outlet holes through one micro-fluidic channel in the prior art is changed, more than two micro-fluidic channels respectively communicated with the sample inlet holes are communicated with the same sample outlet hole, the number of the sample inlet holes and the number of the micro-fluidic channels are increased, the number of the micro-fluidic channels capable of forming cross positioning marks is increased, the distance arrangement can be smaller, and therefore the detection flux of the space omics sequencing method and the mark area of a sample are improved.
According to an embodiment of the present disclosure, the sample section may be an embryonic tissue, a tumor tissue, or the like, which is not limited by the present disclosure.
According to an embodiment of the present disclosure, the pre-processing of the sample slice in step S110 comprises at least one of the following ways:
preparing a sample slice, staining the sample slice, fixing the sample slice, blocking BSA (bovine serum albumin) of the sample slice, performing permeation treatment on the sample slice, and placing the sample slice in a sample area in the middle of a glass slide.
According to an embodiment of the present disclosure, the cross-location identity is determined by different barcode, whose principle is: the microfluidic chip has a sample labeling region (the region having parallel microfluidic channels) overlying the sample slice, and a first set of barcode a's are passed through the microfluidic channels to produce rows parallel to and spatially separated from each other, each row including a1-ANA label, N is a positive integer greater than 1; the microfluidic chip and sample are then washed, spun to place the microfluidic chip perpendicular to the first labeled position, and a second set of barcode B's are passed into the microfluidic channel, thereby creating parallel and spatially separated columns, each comprising B1-BNAnd N is a positive integer greater than 1. After marking, each region of tissue includes a unique composite barcode AiBj(i, j ∈ N) to mark and distinguish different space regions.
According to an embodiment of the present disclosure, the adding a cross-positioning identifier to the sample slice using a microfluidic chip in step S120 includes:
covering the microfluidic chip on the sample slice;
adding first substances containing different barcode A into sample inlet holes of the microfluidic chip respectively, and carrying out a first reaction with the sample slices;
washing the sample slice with a buffer;
rotating the microfluidic chip at a preset angle, so that the direction of a microfluidic channel of the microfluidic chip through which the first substance flows and the direction of the rotated microfluidic channel form the cross positioning mark;
and respectively adding second substances containing different barcode B into the rotated sample inlet holes of the microfluidic chip, and carrying out a second reaction with the sample slice.
According to the embodiment of the disclosure, the microfluidic chip used for performing the second reaction operation and the microfluidic chip for performing the first reaction can be the same chip, and the chip after the first reaction is washed clean is rotated to perform the second reaction. It is understood that, in order to avoid the influence of the residual liquid in the microfluidic channel on the second reaction after the first reaction and the influence on the labeling effect of the sample slice, another new microfluidic chip may be used to operate the second reaction, which is not limited in this disclosure.
The following describes the space omics sequencing methods, specifically space transcriptome sequencing, space proteome sequencing, space epigenome sequencing and space epigenome sequencing, respectively as examples.
Spatial transcriptome sequencing
FIG. 4 is a diagram of the structure of a coding sequence for cross-localization markers in sequencing of the spatial transcriptome, wherein the sequence of a first substance comprising barcode A from the 5 'end to the 3' end is, in order: linker A sequence, barcode A sequence, and multiple T sequence, wherein the multiple T sequence is complementarily combined with the multiple A sequence of mRNA, and the sequence from 5 'end to 3' end of a second substance containing barcode B is: amplification sequence, barcode B sequence, unique molecular identifier sequence (UMI sequence), linker B sequence, wherein the linker A sequence and the linker B sequence are combined in a complementary way or are combined in a complementary way through additional linker sequences, and after labeling, each region of the tissue comprises unique composite barcode AiBjTherefore, different space areas are marked and distinguished.
The first reaction by addition of a first substance comprising barcode a is an inversion reaction and the second reaction by addition of a second substance comprising barcode B is a ligation reaction. Reagents such as RNA barcode A and invertase are added in the first reaction, and reagents such as RNA barcode B ligase are added in the second reaction, which are specifically referred to the prior art, and the details of the disclosure are omitted.
Spatial proteome sequencing
FIG. 5 is a structural diagram of a coding sequence of a cross-localization marker in sequencing of a spatial proteome, which is the same as the coding sequence of the cross-localization marker in sequencing of a spatial transcriptome, and is composed of a first substance containing barcode A and a second substance containing barcode B, which are not repeated herein.
Prior to performing the first reaction, incubating the sample section with a partner coupled to barcode C comprising, from 5 'to 3', a barcode C sequence coupled to the partner and a poly a sequence that complementarily binds to the poly T sequence in the first substance, to allow binding of the partner to the protein of interest on the sample section. The antibody coupled with the barcode C can be introduced into a sample slice through a microfluidic channel to be incubated with the sample slice, or a partner coupled with the barcode C can be added into the sample slice through a pipettor, a full-automatic sample adding workstation and the like to be incubated with tissues in advance, and then the microfluidic chip is covered on the sample slice to carry out a first reaction and a second reaction.
Spatially apparent group sequencing
FIG. 6 is a diagram of the structure of the coding sequences for cross-localization markers in sequencing of the spatial appearance group.
In spatially apparent group sequencing, a partner for a protein of interest and a corresponding buffer are added to a sample section to allow binding of the partner to the protein of interest when the sample section is pretreated. Both the first and second reactions were incubation reactions, with barcode a and barcode B not attached. In the first reaction, the added first substance is pre-embedded PAT mixed liquor with a barcode A joint and corresponding buffer solution and other reagents, and in the second reaction, the added second substance is pre-embedded PAT mixed liquor with a barcode B joint and corresponding buffer solution and other reagents. The PAT mixed solution is a fusion Protein formed by Protein A and transposase Tn5, and has specific antibody targeting property and high-efficiency DNA cutting and linker adding activities, wherein the Protein A can specifically recognize and bind with an Fc segment of an antibody, and the Tn5 can cut DNA efficiently and add a linker sequence. Therefore, the genomic DNA of the tissue can be incubated with PAT to obtain the DNA target fragment added with the specific linker. After a second reaction period, PAT breaks the chromatin in the antibody binding region to obtain target DNA fragments, and EDTA or other reagents capable of chelating metal ions, such as EGTA, etc., with appropriate concentration is added after the reaction is completed to terminate the reaction of PAT. The linker sequence with barcode A or B in PAT is ligated to the target DNA fragment to effect the addition of barcode A and barcode B at both ends of the DNA fragment.
The space omics sequencing method disclosed by the embodiment of the disclosure is applied to space appearance group sequencing, and high-flux space appearance group sequencing is realized by adding barcode A and barcode B at two ends of a DNA fragment.
According to the embodiment of the disclosure, when applied to the sequencing of the spatial appearance group, the method comprises the following steps:
staining of frozen or paraffin tissue sections (or adjacent sections) (optional);
fixation of the slice (optional);
if the section is fixed, performing chromatin opening treatment by using hypotonic buffer solution;
adding a partner of the protein of interest and a corresponding buffer to the section to allow sufficient binding of the partner to the corresponding protein in the tissue;
washing the slices with a wash buffer;
covering the microfluidic chip on the tissue slice, adding a mixed solution of pre-embedded Tn5-barcode A and a reaction buffer solution into the sample injection hole, pumping out the sample injection hole, and incubating for a proper time after filling a liquid in the microfluidic channel; said Tn5-barcode a further comprising partner binding material for binding to said target protein partner;
removing the microfluidic chip and washing the tissue, rotating the chip by 90 degrees, covering the tissue again, adding the mixed solution of pre-embedded Tn5-barcode B and the reaction buffer solution into the sample injection hole, pumping out the sample injection hole, and incubating for a proper time after the microfluidic channel is filled with the liquid; said Tn5-barcode B further comprising partner binding material for binding to said target protein partner;
disrupting chromatin of the sample with Tn5 to form DNA fragments;
EDTA or other reagents capable of chelating metal ions such as EGTA and the like with proper concentration are added into the sample injection hole and pumped out of the sample injection hole, and the sample injection hole is placed at a corresponding temperature for reaction for proper time after the microfluidic channel is filled with liquid so as to stop the reaction.
And dissociating the tissue by using a lysis solution for subsequent cDNA purification and amplification and library construction and sequencing.
In one embodiment, said Tn5-barcode a containing partner binding substance is a fusion protein of partner binding substance and transposase Tn5 pre-embedded barcode a linker; tn5-barcode B containing the partner binding substance is a fusion protein pre-embedded barcode B joint formed by the partner binding substance and transposase Tn 5;
in one embodiment, the partner is selected from an antibody, an antibody Fc fragment; the partner binding substance is selected from protein A, protein G and Fc receptor protein.
Spatially apparent transcriptome sequencing
FIG. 7 is a diagram of a coding sequence structure of a cross-localization marker in sequencing of a spatial appearance transcriptome, which is the same as the coding sequence of the cross-localization marker in sequencing of the spatial transcriptome, and is composed of barcode A and barcode B, and is not repeated herein. It should be noted that, when the sample slice is pretreated, the RNA needs to be inverted by oligo dT or random primer to obtain cDNA, and then the antibody of the target protein and the corresponding buffer are added to the sample slice to combine the antibody with the target protein, which is not repeated herein specifically referring to the technical content of the space appearance group test. In order to avoid the influence of the original DNA in the sample section on the cDNA obtained by reverse transcription, it is also possible to digest the original DNA with DNase I (deoxyribonuclease I) and then reverse the RNA. The space omics sequencing method of the embodiment of the disclosure is applied to space epigenome sequencing, and high-flux space epigenome sequencing is realized by adding barcode A and barcode B at two ends of a cDNA segment.
According to the embodiment of the disclosure, when applied to space appearance transcriptome sequencing, the method comprises the following steps:
staining (optional) of frozen or paraffin tissue sections (or adjacent sections);
fixation of the slice (optional);
digesting the genomic DNA with DNase I (optional);
reverse-converting RNA using oligo dT or random primers;
adding a partner of the protein of interest and a corresponding buffer to the section to allow sufficient binding of the partner to the corresponding protein in the tissue;
washing the slices with a washing buffer;
covering the microfluidic chip on the tissue slice, adding a mixed solution of pre-embedded Tn5-barcode A and a reaction buffer solution into the sample injection hole, pumping out the sample injection hole, and incubating for a proper time after filling a liquid in the microfluidic channel; said Tn5-barcode a further comprising partner binding material for binding to said target protein partner;
removing the microfluidic chip and washing the tissue, rotating the chip by 90 degrees, covering the tissue again, adding the mixed solution of pre-embedded Tn5-barcode B and the reaction buffer solution into the sample injection hole, sucking, and incubating for a proper time after the microfluidic channel is filled with the liquid; said Tn5-barcode B further comprising partner binding material for binding to said target protein partner;
disrupting chromatin of the sample with Tn5 to form DNA fragments;
EDTA or other reagents capable of chelating metal ions such as EGTA and the like with proper concentration are added into the sample inlet and then pumped until the microfluidic channel is filled with liquid, and then the microfluidic channel is placed at a corresponding temperature for reaction for proper time to stop the reaction.
And dissociating the tissue by using a lysis solution for subsequent cDNA purification and amplification and library construction and sequencing.
In one embodiment, said Tn5-barcode a containing partner binding substance is a fusion protein of partner binding substance and transposase Tn5 pre-embedded barcode a linker; the Tn5-barcode B containing the partner binding substance is a fusion protein pre-embedded barcode B joint formed by the partner binding substance and transposase Tn 5;
in one embodiment, the partner is selected from an antibody, an antibody Fc fragment; the partner binding substance is selected from protein A, protein G, Fc receptor protein.
According to an embodiment of the present disclosure, the method of spatial omics sequencing further comprises steps S140-S150.
In step S140, in the process of performing the first reaction and/or the second reaction, the first substance and/or the second substance is smoothly entered into the sample labeling area by using a positive pressure to inflate the sample inlet hole of the microfluidic chip and/or a negative pressure to suck the sample outlet hole of the microfluidic chip; wherein the sample marking area is an area where the microfluidic chip covers the sample slice, and the area is provided with the microfluidic channels which are arranged in parallel;
in the disclosed mode, in order to avoid the blockage of the microfluidic channel, the sample hole can be pumped out under negative pressure, and simultaneously, the sample hole is aerated under positive pressure, so that the added first substance and/or second substance can smoothly enter the sample marking area.
In step S150, a hard object is used to press the microfluidic channel through the anti-backflow hole near the sample outlet hole, so as to prevent the first substance and/or the second substance from flowing back from the sample outlet hole of the microfluidic chip during the first reaction and/or the second reaction.
In the present disclosure, the micro-fluidic channel is deformed by pressing and completely blocks the channel to prevent the material flowing out of the sample outlet from flowing back, and the first material and/or the second material can fully react with the cellular material of the sample slice, and after the reaction is completed, the hard material is taken out, and the pressed micro-fluidic channel can be recovered by itself.
Example 1: the spatial transcriptome sequencing method comprises the following steps:
1. fixation of slices (optional): taking fresh or 7um newborn mouse brain tissue slices stored at-80 ℃, firstly washing with 300ul 1xPBS (phosphate buffer solution) for 10min, and then fixing with 300ul 4% formaldehyde (configured by 1 xPBS) for 20 min;
2. staining of frozen or paraffin tissue sections (or adjacent sections) (optional): and (3) placing the tissue slices in 100% absolute ethyl alcohol for dehydration for 20s, dehydrating in 75% ethyl alcohol for 1.5min, adding 1% cresyl violet staining solution for staining for 2min, instantly washing with 75% ethyl alcohol and 100% absolute ethyl alcohol, airing and photographing.
3. BSA blocking of sections (optional): tissue sections were loaded with 300ul 1% BSA (1xPBS + 1% RNase Inhibitor (RI)) and incubated at room temperature for 30min and washed with 300ul 1xPBS for 3 min.
4. Permeabilization of the sections (optional): adding 300ul of 0.5% TritonX-100 (configured by 1xPBS + 1% RI) to the tissue slices, incubating at room temperature for 10min, washing with 300ul of 1xPBS for 10min, air-drying the tissue slices at room temperature, attaching the tissue slices to the microfluidic chip, and fixing the microfluidic chip with a clamp.
5. Reverse transcription of tissue RNA: separately adding 5ul of an invert solution containing specific RNA barcode a to each well and slowly pumping out of the wells to fill the microfluidic channels with liquid, wherein the invert solution comprises: 1x Maxima H Minus RT buffer, 500uM dNTP, 0.3U/ul Superasein RNase Inhibitor, 0.3U/ul RNase Inhibitor, 0.05xPBS, RNase Free H2O, 20U/ul Maxima H Reverse Transcriptase and 3uM RNA barcode A. Then the mixture is placed at room temperature for reaction for 30min, then placed at 42 ℃ for reaction for 90min, finally the inversion solution is slowly pumped to dryness, 8ul of 1x Neb buffer 3.1+ 0.5% RI is added into each sample hole, and the mixture is slowly pumped to wash for 10 min.
6. Ligation of tissue RNA: the microfluidic chip is rotated by 90 degrees and then covered on the tissue slice again. Adding separately to each well 5ul of a ligation solution containing specific RNA barcode B and slowly pumping out of the wells to fill the microfluidic channels with the solution, wherein the ligation solution comprises: 1x T4 DNA ligase buffer, 0.3U/ul RNase Inhibitor, 0.1U/ul Superasein RNase Inhibitor, 0.1% TritonX-100, RNase Free H2O, 0.5 XNeb buffer 3.1, 16U/ul T4 DNA ligase and 6uM RNA barcode B. And then the mixture is placed at room temperature for reaction for 30min, the rest part of the connecting solution is slowly pumped to be dry, 10ul of 0.1 percent Triton X-100 and 0.5 percent RI are added into each sample inlet hole, and the microfluidic chip is removed after the mixture is slowly pumped and washed for 10 min.
7. Tissue lysis: adding 200ul of a lysis solution to the tissue slices, wherein the lysis solution comprises: 10mM Tris (pH8.0), 200mM NaCl, 50mM EDTA (pH8.0), 2.2% SDS, Water and 1 xPBS. After repeated beating, the lysate was recovered to a 1.5ml EP tube, placed in a 55 ℃ metal bath at 600rpm for lysis for 2h, then removed and stored at-80 ℃.
8. Binding of sample to streptavidin magnetic beads (Myone C1 beads): to 200ul of sample lysate was added 100ul of washed C1 beads and incubated for 30min at room temperature with rotation. Since the 5' end of RNA barcode B carries biotin modification, streptavidin magnetic beads can be used to enrich sample cDNA.
9. Template switching (Template Switch) of sample-bound cDNA: the magnetic beads with sample cDNA bound thereto are first washed. Adding a total of 110ul of prepared template conversion solution into C1 magnetic beads combined with sample cDNA, and uniformly mixing, wherein the template conversion solution comprises: 1 × Maxima H Minus RT Bufer, 1mM dNTP, 1U/ul RNase Inhibitor, RNase Free H2O, 5U/ul Maxima H Reverse Transcriptase Transcriptase and 2.5uM Template Switch Oligo (TSO). Followed by a 30min incubation at room temperature with rotation and finally 1h incubation at 42 ℃ with rotation.
10. Amplification of sample cDNA: adding 120ul of cDNA amplification buffer to the template-converted C1 magnetic beads, wherein the cDNA amplification buffer comprises: 1x Kapa Hifi Master mix, 0.4uM upstream amplification primer, 0.4uM downstream amplification primer and Water. Then evenly mixed, divided into two tubes and placed in a PCR instrument for amplification.
11. Fragment screening of amplified DNA products: to 120ul of the amplified product, 84ul of Kapa Pure Beads were added, mixed well, and allowed to stand for 5 minutes to bind to DNA. Then placed in a magnetic rack, washed 2 times with 200ul 85% ethanol, air dried at room temperature for 3min, and finally eluted the DNA from the beads using 20ul RNase Free Water.
12. Use of the sorted and eluted DNA
Figure BDA0003362653030000151
DNA Library Prep Kit V2 for Illumina reference the trade name product instructions for Library sequencing.
Example 2: the space proteome sequencing method comprises the following steps:
1. fixation of slices (optional): fresh or-80 ℃ stored 7um newborn mouse brain tissue slices are taken, firstly washed for 10min by 300ul 1xPBS, and then fixed for 20min by 300ul 4% formaldehyde (1xPBS configuration);
2. staining of frozen or paraffin tissue sections (or adjacent sections) (optional): and (3) placing the tissue slices in 100% absolute ethyl alcohol for dehydration for 20s, dehydrating in 75% ethyl alcohol for 1.5min, adding 1% cresyl violet staining solution for staining for 2min, instantly washing with 75% ethyl alcohol and 100% absolute ethyl alcohol, airing and photographing.
3. BSA blocking of sections (optional): tissue sections were loaded with 300ul 1% BSA (1xPBS + 1% RNase Inhibitor (RI)) and incubated at room temperature for 30min and washed with 300ul 1xPBS for 3 min.
4. Permeabilization of the sections (optional): adding 300ul 0.5% TritonX-100(1xPBS + 1% RI configuration) to the tissue slices, incubating at room temperature for 10min, washing with 300ul 1xPBS for 10min,
5. incubation of the antibody: 0.1ug of antibody coupled to different barcode was added to the tissue sections and incubated at 4 ℃ for 30min to allow sufficient binding of the antibody to the protein of interest. Washing with 1% BSA + 0.01% Tween 20 wash buffer prepared by 1 × PBS for 3 times and washing with RNase Free Water for 1 time, after the tissue slice is dried at room temperature, attaching the tissue slice to the microfluidic chip, and fixing the tissue slice with a clamp;
6. reverse transcription of antibody barcode C: separately, 5ul of an invert solution containing specific RNA barcode a was added to each well and slowly aspirated out of the wells to fill the microfluidic channels with liquid, wherein the invert solution comprises: 1x Maxima H Minus RT buffer, 500uM dNTP, 0.3U/ul Superasein RNase Inhibitor, 0.3U/ul RNase Inhibitor, 0.05xPBS, RNase Free H2O, 20U/ul Maxima H Reverse Transcriptase and 3uM RNA barcode A. Then the mixture is placed at room temperature for reaction for 30min, then placed at 42 ℃ for reaction for 90min, finally the inversion solution is slowly pumped to dryness, 8ul 1x Neb buffer 3.1+ 0.5% RI is added into each sample hole, and the mixture is slowly pumped and washed for 10 min.
7. Ligation of antibody barcode C: the microfluidic chip is rotated by 90 degrees and then covered on the tissue section again. Adding separately to each well 5ul of a ligation solution containing specific RNA barcode B and slowly pumping out of the wells to fill the microfluidic channels with the solution, wherein the ligation solution comprises: 1x T4 DNA ligase buffer, 0.3U/ul RNase Inhibitor, 0.1U/ul Superasein RNase Inhibitor, 0.1% TritonX-100, RNase Free H2O, 0.5 XNeb buffer 3.1, 16U/ul T4 DNA ligase and 6uM RNA barcode B. And then the mixture is placed at room temperature for reaction for 30min, the rest part of the connecting solution is slowly pumped to be dry, 10ul of 0.1 percent Triton X-100 and 0.5 percent RI are added into each sample inlet hole, and the microfluidic chip is removed after the mixture is slowly pumped and washed for 10 min.
8. Tissue lysis: adding 200ul of a lysis solution to the tissue slices, wherein the lysis solution comprises: 10mM Tris (pH8.0), 200mM NaCl, 50mM EDTA (pH8.0), 2.2% SDS, Water and 1 xPBS. After repeated beating, the lysate was recovered to a 1.5ml EP tube, placed in a 55 ℃ metal bath at 600rpm for lysis for 2h, then removed and stored at-80 ℃.
9. Binding of sample to streptavidin magnetic beads (Myone C1 beads): to 200ul of sample lysate, 100ul of washed C1 beads was added and incubated for 30min at room temperature with rotation. Since the 5' end of RNA barcode B carries biotin modification, streptavidin magnetic beads can be used to enrich sample cDNA.
10. Template switching (Template Switch) for binding antibody barcode C: the magnetic beads bound to antibody barcode C are first washed. Adding the prepared total 110ul template conversion solution into C1 magnetic beads combined with antibody barcode C, and uniformly mixing, wherein the template conversion solution comprises: 1 × Maxima H Minus RT Bufer, 1mM dNTP, 1U/ul RNase Inhibitor, RNase Free H2O, 5U/ul Maxima H Reverse Transcriptase Transcriptase and 2.5uM Template Switch Oligo (TSO). Followed by a 30min incubation at room temperature with rotation and finally 1h incubation at 42 ℃ with rotation.
11. Amplification of antibody barcode C: adding 120ul of amplification buffer to the template-converted C1 magnetic beads, wherein the amplification buffer comprises: 1x Kapa Hifi Master mix, 0.4uM upstream amplification primer, 0.4uM downstream amplification primer and Water. Then evenly mixed, divided into two tubes and placed in a PCR instrument for amplification.
12. Fragment screening of amplified DNA products: to 120ul of the amplified product, 84ul of Kapa Pure Beads were added, mixed well, and allowed to stand for 5 minutes to bind to DNA. Then placed in a magnetic rack, the supernatant was aspirated into a new EP tube, 108ul Kapa Pure Beads were added, and left for 5 minutes to bind to DNA. Then placed on a magnetic rack, the supernatant was discarded, washed 2 times with 200ul 85% ethanol, air-dried at room temperature for 3min, and finally the DNA was eluted from the beads using 20ul RNase Free Water.
13. And (3) carrying out library construction sequencing on the sorted and eluted DNA by using a related library construction kit according to the product instruction of a merchant.
Example two
Fig. 8 shows a schematic flow diagram of a method of location identification on a slide according to an embodiment of the present disclosure. As shown in fig. 8, the spatial omics sequencing method comprises steps S210-S240.
In step S210, a slide is subjected to pretreatment;
in step S210, adding a first substance containing barcode a to a sample inlet of the microfluidic chip, and performing a first reaction with the glass slide;
in step S230, rotating the microfluidic chip by a predetermined angle, so that the direction of the microfluidic channel of the microfluidic chip through which the first substance flows and the rotated direction of the microfluidic channel form the cross-location mark;
in step S240, adding a second substance containing barcode B to the rotated sample inlet of the microfluidic chip, and performing a second reaction with the first substance performing the first reaction on the slide glass; wherein the barcode A and barcode B are used to mark the cross-location marker formed on the slide glass.
According to the positioning identification method on the glass slide, a chip with various barcode combinations is obtained by connecting barcode A and barcode B on the glass slide, and the manufactured chip can be placed at-80 ℃ for later use and can be specifically applied to sequencing aspects such as space transcriptome, space proteome, space appearance transcriptome and space appearance transcriptome.
According to an embodiment of the present disclosure, the pre-processing the slide comprises: coating one of gold nanoparticles, streptavidin or amino modification on a sample area of the glass slide, so that the modified area covers the sample area.
According to the embodiment of the disclosure, the first substance may be a mixed solution containing RNA barcode a and a nucleic acid coupling agent, etc. to couple the RNA barcode a and the glass slide, wherein, if the nano-gold particles are modified on the glass slide, the 5 'end of the RNA barcode a may be subjected to thiol modification, and if the nano-gold particles are modified on the glass slide, the 5' end of the RNA barcode a may be subjected to carboxyl modification; if streptavidin is modified on the slide, the 5' end of RNA barcode A can be biotin modified. The second substance may be a mixture liquid containing RNA barcode B and ligase or the like that have been annealed beforehand with a linker connecting sequence, so that RNA barcode B and RNA barcode A are ligated with the aid of the linker connecting sequence and ligase.
Through the modification, the barcode can be stably fixed on the glass slide, and the marking loss caused by flushing the barcode by washing liquid in the washing process is avoided; more barcode is marked on the glass slide through the interaction of streptomycin and biotin, and the marking flux is improved.
According to an embodiment of the present disclosure, the method of location identification on a slide further includes steps S250-S260.
In step S250, in the process of performing the first reaction and/or the second reaction, the first substance and/or the second substance is/are smoothly entered into the slide mark area by using a negative pressure to suck the sample outlet hole of the microfluidic chip; the glass slide marking area is a central area of the glass slide covered by the microfluidic chip and is provided with microfluidic channels arranged in parallel;
in the disclosed mode, in order to avoid the blockage of the microfluidic channel, the sample hole can be pumped out under negative pressure, and simultaneously, the sample hole is aerated under positive pressure, so that the added first substance and/or second substance can smoothly enter the sample marking area.
In step S260, a hard object is used to penetrate through the anti-backflow hole near the sample outlet hole to press the microfluidic channel, so as to prevent the first substance and/or the second substance from flowing back from the sample outlet hole of the microfluidic chip during the first reaction and/or the second reaction.
In the present disclosure, the micro-fluidic channel is deformed by pressing and completely blocks the channel to prevent the material flowing out of the sample outlet from flowing back, and the first material and/or the second material can fully react with the cellular material of the sample slice, and after the reaction is completed, the hard material is taken out, and the pressed micro-fluidic channel can be recovered by itself.
The following describes a specific experimental procedure for locating the markers on the slide:
1. coating nano gold particles and streptavidin on a sample area of the glass slide or performing amino modification on the sample area of the glass slide;
2. covering the microfluidic chip on a glass slide, adding sulfhydryl, biotin or carboxyl modified barcode A into the sample inlet, and coupling the sample inlet with the glass slide through nanogold (sulfhydryl modification), streptavidin (biotin modification) or amino modification (carboxyl modification);
3. washing to remove excess unbound barcode a;
4. rotating the micro-fluidic chip by 90 degrees, covering the micro-fluidic chip on the glass slide again, and adding a mixed solution containing barcode B and T4 ligase into the sample inlet to connect barcode B with barcode A;
5. washing to remove excess unbound barcode B;
6. the prepared barcode chip was placed at-80 ℃ for use.
EXAMPLE III
Figure 9 shows a schematic flow diagram of a single cell epigenetics sequencing method according to an embodiment of the disclosure. As shown in fig. 9, the epigenomic sequencing method includes steps S310-S330.
In step S310, a sample is pretreated;
in step S320, adding a target protein partner to the pretreated sample, and allowing the target protein partner to bind to the target protein;
in step S330, the sample is subjected to an incubation reaction with a first substance comprising barcode a and a second substance comprising barcode B, the first and second substances further comprising Tn5 and a target protein partner binding substance for binding to the target protein partner, the Tn5 disrupting the cDNA of the sample to form a cDNA fragment, while barcode a and barcode B are added to the cDNA fragment, followed by cell lysis, cDNA amplification and library sequencing in sequence.
The sequencing of the epigenetics provided by the embodiment of the disclosure can be applied to single cell sequencing, and can also be applied to space epigenetics sequencing by combining with a microfluidic chip, and the detection of the single cell epigenetics of a tissue sample is realized by adding a barcode A and a barcode B at two ends of a cDNA fragment.
According to an embodiment of the present disclosure, the pre-processing the sample in step S310 includes:
immobilization of cells in the sample;
reversing the RNA in the sample using primers;
and optionally a step of digesting the genomic DNA with a digestion reagent after immobilization.
According to the embodiment of the disclosure, when the method is applied to single-cell epigenet transcriptome space omics sequencing, the method comprises the following steps:
fixing the cells on a slide;
optionally comprising the step of digesting the genomic DNA with a digestion reagent;
reversing the RNA of the cells using primers;
adding a partner of the protein of interest to the pretreated cells, such that the partner of the protein of interest is substantially bound to the protein of interest;
washing the cells with a wash buffer;
adding a mixed solution of pre-embedded Tn5-barcode A and Tn5-barcode B and a reaction buffer solution into the cells for incubation; said Tn5-barcode a, Tn5-barcode B further comprising partner binding material for binding to said target protein partner;
the Tn5 breaks the chromatin of the sample to form DNA fragments, EDTA or other reagents which can chelate metal ions such as EGTA with proper concentration is added after the reaction is finished to stop the reaction, and simultaneously, barcode A and barcode B are added on the DNA fragments, and then the lysis solution is used for sequentially carrying out cell lysis, cDNA amplification and library construction sequencing.
In one embodiment, said Tn5-barcode a containing partner binding substance is a fusion protein of partner binding substance and transposase Tn5 pre-embedded barcode a linker; the Tn5-barcode B containing the partner binding substance is a fusion protein pre-embedded barcode B joint formed by the partner binding substance and transposase Tn 5;
in one embodiment, the partner is selected from an antibody, an antibody Fc fragment; the partner binding substance is selected from protein A, protein G and Fc receptor protein.
The foregoing description is only exemplary of the preferred embodiments of the disclosure and is illustrative of the principles of the technology employed. It will be appreciated by those skilled in the art that the scope of the invention in the present disclosure is not limited to the specific combination of the above-mentioned features, but also encompasses other embodiments in which any combination of the above-mentioned features or their equivalents is possible without departing from the inventive concept. For example, the above features and (but not limited to) the features disclosed in this disclosure having similar functions are replaced with each other to form the technical solution.

Claims (10)

1. A method of sequencing a space group, comprising:
pretreating a sample slice;
covering the microfluidic chip on the sample slice;
adding first substances containing different barcode A into sample inlet holes of the microfluidic chip respectively, and carrying out a first reaction with the sample slices;
washing the sample slice with a buffer;
rotating the microfluidic chip at a preset angle, so that the direction of a microfluidic channel of the microfluidic chip through which the first substance flows and the direction of the rotated microfluidic channel form the cross positioning mark;
adding second substances containing different barcode B into the sample inlet holes of the rotated microfluidic chip respectively, and performing a second reaction with the sample slice; wherein the first and second substances further comprise Tn5, and the Tn5 breaks the chromatin in the partner binding region to obtain a target DNA fragment, and barcode A and barcode B are added to the target DNA fragment;
and sequentially carrying out cell lysis, amplification and library establishment on the sample slices, and then carrying out gene sequencing according to the cross-localization markers.
2. The method for spatial omic sequencing of claim 1, wherein prior to the first reaction, a partner for the protein of interest is added to bind to the protein of interest; optionally, a step of reverse treatment of RNA with primers to obtain cDNA is also included before adding the partner.
3. The method of spacemic sequencing of claim 2 further comprising:
the DNA of the sample sections is digested with a digestion reagent before RNA inversion treatment.
4. A method of sequencing a space group, comprising:
pretreating a sample slice;
adding a partner of the target protein to the sample section;
covering the microfluidic chip on the sample slice, adding a mixed solution of pre-embedded Tn5-barcode A into the sample inlet, pumping out the sample inlet, and carrying out a first reaction after the microfluidic channel is filled with liquid; said mixed solution of Tn5-barcode a further comprises a partner binding substance for binding a partner of said target protein;
removing the microfluidic chip, washing the sample slice, rotating the microfluidic chip by 90 degrees, covering the microfluidic chip on the sample slice again, adding the mixed solution of pre-embedded Tn5-barcode B into the sample inlet, pumping out the sample inlet, and carrying out a second reaction after the microfluidic channel is filled with liquid; said mixed solution of Tn5-barcode B further comprises a partner binding substance for binding a partner of said target protein;
breaking the chromatin of the sample slice with Tn5 to form DNA fragments while adding barcode A and barcode B on the DNA fragments;
adding EDTA or EGTA into the sample inlet, pumping out the sample inlet until the microfluidic channel is filled with liquid, and reacting at corresponding temperature for a proper time to terminate the reaction;
and dissociating the sample slice by using the lysis solution for subsequent cDNA purification and amplification and library construction sequencing.
5. The method for sequencing according to claim 4, wherein the method further comprises the step of inverting the RNA with primers to obtain cDNA prior to adding the binding partners.
6. The method of spatially omic sequencing of claim 5, further comprising:
the DNA of the sample sections is digested with a digestion reagent before RNA inversion treatment.
7. The method for spatial omic sequencing of claim 4, wherein said partner is selected from the group consisting of an antibody, an antibody Fc fragment; the partner binding substance is selected from protein A, protein G, Fc receptor protein.
8. The method for spatially omics sequencing of any one of claims 1 to 7, wherein the pretreatment of the sample slices comprises at least one of the following:
preparing a sample slice, staining the sample slice, fixing the sample slice, blocking BSA (bovine serum albumin) of the sample slice, performing permeation treatment on the sample slice, and placing the sample slice in a sample area in the middle of a glass slide.
9. The spacemic sequencing method of any of claims 1-7 wherein the number of wells of the microfluidic chip is less than the number of wells to increase the number of wells and microfluidic channels forming the cross-location identifier.
10. The method for sequencing spatially integrated as claimed in claim 9, wherein during the first reaction and/or the second reaction, the first substance and/or the second substance is smoothly introduced into the sample labeling zone by way of positive pressure aeration of the sample inlet holes of the microfluidic chip and/or negative pressure suction of the sample outlet holes of the microfluidic chip; wherein the sample marking area is an area where the microfluidic chip covers the sample slice, and the area is provided with the microfluidic channels which are arranged in parallel; and/or in the process of carrying out the first reaction and/or the second reaction, a hard object penetrates through the anti-backflow hole near the sample outlet hole of the chip to extrude the microfluidic channel, so that the microfluidic channel deforms and completely blocks the channel; and after the reaction is finished, taking out the hard object, and recovering the extruded microfluidic channel by self.
CN202111372033.5A 2020-09-29 2020-09-29 Space omics sequencing method Pending CN114085897A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202111372033.5A CN114085897A (en) 2020-09-29 2020-09-29 Space omics sequencing method

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
CN202111372033.5A CN114085897A (en) 2020-09-29 2020-09-29 Space omics sequencing method
CN202011057632.3A CN112143784A (en) 2020-09-29 2020-09-29 Space omics sequencing, single-cell apparent transcriptomics sequencing and positioning identification method

Related Parent Applications (1)

Application Number Title Priority Date Filing Date
CN202011057632.3A Division CN112143784A (en) 2020-09-29 2020-09-29 Space omics sequencing, single-cell apparent transcriptomics sequencing and positioning identification method

Publications (1)

Publication Number Publication Date
CN114085897A true CN114085897A (en) 2022-02-25

Family

ID=73894361

Family Applications (3)

Application Number Title Priority Date Filing Date
CN202111372042.4A Pending CN114045332A (en) 2020-09-29 2020-09-29 Single cell epigenetics sequencing method
CN202111372033.5A Pending CN114085897A (en) 2020-09-29 2020-09-29 Space omics sequencing method
CN202011057632.3A Pending CN112143784A (en) 2020-09-29 2020-09-29 Space omics sequencing, single-cell apparent transcriptomics sequencing and positioning identification method

Family Applications Before (1)

Application Number Title Priority Date Filing Date
CN202111372042.4A Pending CN114045332A (en) 2020-09-29 2020-09-29 Single cell epigenetics sequencing method

Family Applications After (1)

Application Number Title Priority Date Filing Date
CN202011057632.3A Pending CN112143784A (en) 2020-09-29 2020-09-29 Space omics sequencing, single-cell apparent transcriptomics sequencing and positioning identification method

Country Status (1)

Country Link
CN (3) CN114045332A (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891858A (en) * 2022-07-13 2022-08-12 广州国家实验室 Chromatin three-dimensional conformation capture method and application thereof

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2022067565A1 (en) * 2020-09-29 2022-04-07 生物岛实验室 Spatial omics sequencing method, single cell epitranscriptomic sequencing method, and localization labeling method
CN113270142A (en) * 2021-05-19 2021-08-17 东南大学 Space transcriptome sequencing decoding method based on transient coding
CN113930847A (en) * 2021-09-26 2022-01-14 东南大学 Space transcriptome position information coding chip and preparation method and application thereof
CN114231607A (en) * 2021-10-26 2022-03-25 吉林大学 Microarray chip and preparation method and application thereof
WO2023159416A1 (en) * 2022-02-24 2023-08-31 浙江大学 Single-cell transcriptome sequencing method and use thereof

Citations (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN204865880U (en) * 2015-08-25 2015-12-16 辽宁中医药大学 A micro -fluidic chip for having more screening of material compatibility drug effect
CN105473744A (en) * 2013-06-25 2016-04-06 普罗格诺西斯生物科学公司 Spatially encoded biological assays using a microfluidic device
CN106591105A (en) * 2016-12-09 2017-04-26 东北大学 Microorganism automatic sample introduction system based on microfluidic chip and preparation method thereof
CN106925359A (en) * 2017-04-21 2017-07-07 苏州汶颢微流控技术股份有限公司 Micro grade is than loading micro-fluidic chip system and upper quadrat method
CN107597219A (en) * 2017-09-13 2018-01-19 北京理工大学 A kind of micro-fluidic chip sample introduction and its bonding techniques
CN108315387A (en) * 2018-02-07 2018-07-24 北京大学 Few cells ChIP methods
CN108779492A (en) * 2015-12-30 2018-11-09 生物辐射实验室股份有限公司 Digital quantification of protein
US20190338279A1 (en) * 2016-12-07 2019-11-07 Mgi Tech Co., Ltd. Method for constructing single cell sequencing library and use thereof
GB201918340D0 (en) * 2019-12-12 2020-01-29 Cambridge Entpr Ltd Spatial barcoding

Family Cites Families (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN203842597U (en) * 2014-05-26 2014-09-24 博奥生物集团有限公司 Micro-fluidic chip with deformable membrane capable of preventing reflux
CN107583676B (en) * 2016-07-07 2020-03-06 大连医科大学 Micro-fluidic chip and research method for capturing and detecting exosome
EP3580354A4 (en) * 2017-02-13 2021-03-31 Yale University High-throughput single-cell polyomics
CN206881732U (en) * 2017-04-21 2018-01-16 苏州汶颢微流控技术股份有限公司 Micro grade is than loading micro-fluidic chip system
CN111655848A (en) * 2017-11-21 2020-09-11 阿瑞玛基因组学公司 Preserving spatial proximity and molecular proximity in nucleic acid templates
US20210332425A1 (en) * 2018-08-28 2021-10-28 Rajiv Bharadwaj Increasing spatial array resolution
EP3894591A2 (en) * 2018-12-10 2021-10-20 10X Genomics, Inc. Imaging system hardware
CN111549099B (en) * 2020-04-23 2021-07-30 生物岛实验室 Third-generation sequencing-based single-cell transcriptome sequencing method
CN111676279A (en) * 2020-05-20 2020-09-18 东南大学 Spatial transcription set construction method

Patent Citations (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN105473744A (en) * 2013-06-25 2016-04-06 普罗格诺西斯生物科学公司 Spatially encoded biological assays using a microfluidic device
CN204865880U (en) * 2015-08-25 2015-12-16 辽宁中医药大学 A micro -fluidic chip for having more screening of material compatibility drug effect
CN108779492A (en) * 2015-12-30 2018-11-09 生物辐射实验室股份有限公司 Digital quantification of protein
US20190338279A1 (en) * 2016-12-07 2019-11-07 Mgi Tech Co., Ltd. Method for constructing single cell sequencing library and use thereof
CN106591105A (en) * 2016-12-09 2017-04-26 东北大学 Microorganism automatic sample introduction system based on microfluidic chip and preparation method thereof
CN106925359A (en) * 2017-04-21 2017-07-07 苏州汶颢微流控技术股份有限公司 Micro grade is than loading micro-fluidic chip system and upper quadrat method
CN107597219A (en) * 2017-09-13 2018-01-19 北京理工大学 A kind of micro-fluidic chip sample introduction and its bonding techniques
CN108315387A (en) * 2018-02-07 2018-07-24 北京大学 Few cells ChIP methods
GB201918340D0 (en) * 2019-12-12 2020-01-29 Cambridge Entpr Ltd Spatial barcoding
WO2021116715A1 (en) * 2019-12-12 2021-06-17 Cancer Research Technology Limited Spatial barcoding

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JASON D BUENROSTRO: "Single-cell chromatin accessibility reveals principles of regulatory variation", 《NATURE》 *
YANG LIU ET AL.: "High-Spatial-Resolution Multi-Omics Atlas Sequencing of Mouse Embryos via Deterministic Barcoding in Tissue", 《BIORXIV》 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN114891858A (en) * 2022-07-13 2022-08-12 广州国家实验室 Chromatin three-dimensional conformation capture method and application thereof

Also Published As

Publication number Publication date
CN112143784A (en) 2020-12-29
CN114045332A (en) 2022-02-15

Similar Documents

Publication Publication Date Title
CN114085897A (en) Space omics sequencing method
US20220119869A1 (en) Methods for determining a location of an analyte in a biological sample
JP2022549505A (en) Deterministic barcoding for spatial omics sequence analysis
US11566275B2 (en) Chromatin immunocapture devices and methods of use
CN107709574B (en) Spatial mapping of molecular profiles of biological tissue samples
US20210237022A1 (en) Capturing oligonucleotides in spatial transcriptomics
CN110520541B (en) High throughput single cell multicellular multigenetics
Park et al. Selection and elution of aptamers using nanoporous sol-gel arrays with integrated microheaters
CN116034166A (en) Spatial analysis of DNA methylation
WO2020047007A4 (en) Methods for generating spatially barcoded arrays
WO2016037358A1 (en) Isolated oligonucleotide and use thereof in nucleic acid sequencing
FI3583214T3 (en) Methods and compositions for identifying or quantifying targets in a biological sample
WO2012047015A2 (en) Multiplex microfluidic device for selecting nucleic acid aptamers, and high throughput selection method for nucleic acid aptamers using same
EP3619319B1 (en) Single cell analysis
WO2015009967A1 (en) Digital assay for quantifying and concentrating analytes
WO2022067565A1 (en) Spatial omics sequencing method, single cell epitranscriptomic sequencing method, and localization labeling method
JP7049103B2 (en) Comprehensive 3'end gene expression analysis method for single cells
WO2022135598A1 (en) Biochip for spatial transcriptomic analysis, manufacturing method therefor and application thereof
US20160367986A1 (en) Microfluidic devices and arrangements for introducing reagents and biological samples to such devices
CN111801428A (en) Method for obtaining single-cell mRNA sequence
AU2021442183A1 (en) Construction method for rna sequencing library, sequencing method, and kit
CN116162538B (en) Microfluidic chip and kit for simultaneously detecting protein and RNA
WO2023116938A1 (en) Biochip for spatial transcriptomic analysis, and preparation method and application thereof
CN112986573B (en) Quantitative detection method for exosome multi-group chemical markers
WO2019116306A1 (en) System for capture or immunoprecipitation of a protein-dna complex

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication
RJ01 Rejection of invention patent application after publication

Application publication date: 20220225