CN114045332A - Single cell epigenetics sequencing method - Google Patents
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- CN114045332A CN114045332A CN202111372042.4A CN202111372042A CN114045332A CN 114045332 A CN114045332 A CN 114045332A CN 202111372042 A CN202111372042 A CN 202111372042A CN 114045332 A CN114045332 A CN 114045332A
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Abstract
The embodiment of the disclosure discloses a single cell epigenetics sequencing method, which is characterized by comprising the following steps: pretreating a sample; adding a target protein partner to the pretreated sample, and allowing the target protein partner to bind to the target protein; subjecting said sample to an incubation reaction with a first material comprising barcode a and a second material comprising barcode B, said first and second materials further comprising Tn5 and a target protein partner binding material for binding to said target protein partner, said Tn5 disrupting cDNA of said sample to form a cDNA fragment, while adding barcode a and barcode B to said cDNA fragment, followed by sequentially performing cell lysis, cDNA amplification and library sequencing.
Description
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 single cell epigenetics 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 address the problems in the related art, embodiments of the present disclosure provide a single cell epigenetics sequencing method.
The embodiment of the disclosure provides a single cell epigenetics sequencing method.
Specifically, the single cell epigenetics sequencing method comprises the following steps:
pretreating a sample;
adding a target protein partner to the pretreated sample, and allowing the target protein partner to bind to the target protein;
subjecting said sample to an incubation reaction with a first material comprising barcode a and a second material comprising barcode B, said first and second materials further comprising Tn5 and a target protein partner binding material for binding to said target protein partner, said Tn5 disrupting cDNA of said sample to form a cDNA fragment, while adding barcode a and barcode B to said cDNA fragment, followed by sequentially performing cell lysis, cDNA amplification and library sequencing.
Optionally, the pre-treating the sample comprises:
immobilization of cells in the sample;
reversing the RNA in the sample using primers;
and optionally a step of digesting the DNA of said sample with a digestion reagent after fixation.
Optionally, the target protein partner is selected from an antibody, an antibody Fc fragment; the target protein partner binding substance is selected from protein A, protein G, Fc receptor protein.
The technical scheme provided by the embodiment of the disclosure can have the following beneficial effects:
the sequencing method of the single cell epigenetics of the embodiment of the disclosure preprocesses a sample; adding a target protein partner to the pretreated sample, and allowing the target protein partner to bind to the target protein; incubating the sample 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 cDNA of the sample to form a cDNA fragment, while adding barcode a and barcode B to the cDNA fragment, followed by sequentially performing cell lysis, cDNA amplification and library sequencing, thereby achieving single cell epigenome detection of the tissue sample.
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 of the cross-localization markers in the 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 marking region 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 substance is removed and the squeezed 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 384 sample wells 113 can be arranged on a chip area of 11cm × 11cm, so that 147456 label combinations of 384 × 384 can be realized, the effective labeling rate for a 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 768x 768, 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 barcode at the marked cell position, has simple operation steps, is not limited to space transcriptome sequencing, space proteome sequencing, space appearance set sequencing, space appearance transcriptome 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 the coding sequence of the cross-localization marker in the sequencing of the space transcriptome, wherein the sequence of the first substance containing barcode A from the 5 'end to the 3' end is: 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: an amplification sequence, a barcode B sequence, a unique molecular identifier sequence (UMI sequence), a linker B sequence, said linker A sequence being complementarily bound to the linker B sequence, or said linker A sequence, linker B sequence being linked to one another by an additional linker sequenceComplementary binding, marked, each region of tissue comprising a unique composite barcode AiBjTherefore, different space regions 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 space proteome sequencing, which is the same as the coding sequence of the cross-localization marker in space transcriptome sequencing, and consists of a first substance containing barcode A and a second substance containing barcode B, and is 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 (optional) of frozen or paraffin tissue sections (or adjacent sections);
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; 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.
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 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, 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 for spatial omic 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 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.
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 300ul1xPBS (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 300ul1xPBS 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 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.
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 T4DNA 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 T4DNA 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 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
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 300ul1xPBS, 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 300ul1xPBS 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 300ul1xPBS 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 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 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 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 T4DNA 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 T4DNA 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 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.
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 pretreated;
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-treating 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 positioning 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 epigenetics sequencing method comprises 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 sequentially performing cell lysis, cDNA amplification and library sequencing.
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 digesting agent;
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, 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 (3)
1. A method for single cell epigenetics sequencing, comprising:
pretreating a sample;
adding a target protein partner to the pretreated sample, and allowing the target protein partner to bind to the target protein;
subjecting said sample to an incubation reaction with a first material comprising barcode a and a second material comprising barcode B, said first and second materials further comprising Tn5 and a target protein partner binding material for binding to said target protein partner, said Tn5 disrupting cDNA of said sample to form a cDNA fragment, while adding barcode a and barcode B to said cDNA fragment, followed by sequentially performing cell lysis, cDNA amplification and library sequencing.
2. The method of claim 1, wherein the pre-treating the sample comprises:
immobilization of cells in the sample;
reversing the RNA in the sample using primers;
and optionally a step of digesting the DNA of said sample with a digestion reagent after fixation.
3. The method of claim 1 or 2, wherein the target protein partner is selected from the group consisting of an antibody, an antibody Fc fragment; the target protein partner binding substance is selected from protein A, protein G, Fc receptor protein.
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| CN113270142B (en) * | 2021-05-19 | 2024-09-24 | 东南大学 | Space transcriptome sequencing decoding method based on transient coding |
| CN113930847B (en) * | 2021-09-26 | 2024-05-28 | 东南大学 | Space transcriptome position information coding chip and preparation method and application thereof |
| CN114231607B (en) * | 2021-10-26 | 2024-06-11 | 江西烈冰生物科技有限公司 | Micro-lattice chip and preparation method and application thereof |
| CN115109149B (en) * | 2022-06-21 | 2024-08-09 | 厦门大学 | Screening method of monoclonal antibody |
| CN114891858B (en) * | 2022-07-13 | 2023-05-02 | 广州国家实验室 | Chromatin three-dimensional conformation capturing method and application thereof |
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