WO2022067565A1 - 空间组学测序、单细胞表观转录组学测序及定位标识方法 - Google Patents
空间组学测序、单细胞表观转录组学测序及定位标识方法 Download PDFInfo
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- 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
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- 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
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Definitions
- the present disclosure relates to the technical field of gene sequencing, in particular to a method for spatial omics sequencing, single-cell epitranscriptomic sequencing and localization identification.
- Spatial heterogeneity is a key feature of organ function, and the location information of cells is very important for the study of cell regulatory mechanisms and cell lineage processes.
- Traditional genetic sequencing is performed on a tissue sample or cell population as a whole, and differences between cells may be masked by averaging.
- Single-cell testing techniques can reveal the genetic information of each cell at the single-cell level, enabling different cell types distinguish.
- the ability to reveal the original location information of cells in tissues is still lacking. Therefore, there is a need for a sequencing method that preserves the original spatial location information of cells.
- the embodiments of the present disclosure provide a method for spatial omics sequencing, single-cell epitranscriptomic sequencing, and localization identification.
- the embodiments of the present disclosure provide a spatial omics sequencing method.
- the spatial omics sequencing method includes:
- sample slices are sequentially subjected to cell lysis, amplification and library building, gene sequencing is performed according to the cross-localization identification.
- a microfluidic chip to add cross-positioning marks to the sample slices includes:
- Second substances containing different barcode B are respectively added to the sample injection holes of the rotated microfluidic chip, and a second reaction is carried out with the sample slices.
- the method further includes:
- the method of inflating the sample inlet hole of the microfluidic chip with positive pressure and/or suctioning the sample outlet hole of the microfluidic chip with negative pressure to promote the first substance and/or the second substance to smoothly enter the sample marking area; wherein, the sample marking area is the area where the microfluidic chip covers the sample slice, and this area has the microfluidics arranged in parallel. and/or squeeze the microfluidic channel by using a hard object to pass through the anti-reflux hole near the sample outlet to prevent the first substance from being damaged during the first reaction and/or the second reaction. And/or the second substance flows back from the sample outlet hole of the microfluidic chip.
- the method is applied to spatial transcriptome sequencing or spatial proteome sequencing, wherein the first reaction is an inversion reaction, and the second reaction is a ligation reaction, to add barcode A on the mRNA of the sample slice. and barcode B.
- the method is applied to spatial proteome sequencing, wherein, before the first reaction, a partner coupled with barcode C is added to bind the target protein of interest, and then the first reaction and the second reaction are sequentially performed. reaction.
- the method is applied to spatial epigroup sequencing, wherein the first substance and the second substance further comprise Tn5, and the Tn5 interrupts the chromatin of the partner binding region to obtain the target DNA fragment, and at the same time in the Add barcode A and barcode B to the DNA fragment.
- the method is applied to spatial epitranscriptome sequencing, wherein, prior to the first reaction, a partner of the protein of interest is added to bind the protein of interest; optionally, prior to the addition of the partner, the use of Steps in which primers reverse RNA to obtain cDNA.
- the method further includes:
- the DNA of the sample section is digested with a digestion reagent prior to RNA inversion processing.
- preprocessing sample slices includes at least one of the following:
- Preparation of sample slices Preparation of sample slices, staining of sample slices, fixation of sample slices, BSA blocking of sample slices, permeabilization of sample slices, placing sample slices in the sample area in the middle of the glass slide.
- an embodiment of the present disclosure provides a method for positioning and marking on a glass slide.
- the method for positioning and marking on the glass slide includes:
- the barcode A and the barcode B are used to mark the cross positioning mark formed on the glass slide.
- the pretreatment of the glass slide includes: coating the sample area in the middle of the glass slide with nano gold particles or streptavidin or performing amino modification.
- the first substance and/or the second The substance enters the glass slide marking area smoothly; wherein, the glass slide marking area is the central area of the microfluidic chip covering the glass slide, and this area has microfluidic channels arranged in parallel; and/or
- the method is applied to spatial transcriptome sequencing, spatial proteome sequencing, spatial epigenetic sequencing, and spatial epitranscriptome sequencing.
- the embodiments of the present disclosure provide a single-cell epitranscriptomic sequencing method.
- the single-cell epitranscriptomic sequencing method includes:
- the first substance and the second substance also include Tn5 and a target protein partner binding substance, the target protein partner
- the binding substance is used to bind the target protein partner, the Tn5 interrupts the chromatin of the sample to form a DNA fragment, and at the same time adds barcode A and barcode B to the DNA fragment, and then sequentially performs cell lysis and cDNA amplification and library sequencing.
- the pretreatment of the sample includes:
- a substance including barcode is added from the injection hole of the microfluidic chip to form a cross localization mark marking the spatial position of the cell, and then the cross localization is performed.
- Cell material such as RNA, DNA, or protein
- the above sequencing method uses a microfluidic chip to add substances including different barcodes that mark the position of the cells, the operation steps are simple, and are not limited to spatial transcriptome sequencing, spatial proteome sequencing, spatial epigenetic sequencing, and spatial epitranscriptome sequencing, etc., Wide range of applications.
- the number of sample outlet holes is less than the number of sample inlet holes, which changes the previous design of a microfluidic channel connecting the sample inlet hole and the sample outlet hole, so that more than two sample holes are connected to the sample hole separately.
- the connected microfluidic channels are connected to the same sample outlet, which increases the number of injection holes and microfluidic channels, and increases the number of microfluidic channels that can form cross-positioning marks, which can further reduce the spacing and width of the microfluidic channels, thereby increasing the number of microfluidic channels.
- the detection throughput of the spatial omics sequencing method and the labeling area of the sample were analyzed.
- 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
- FIG. 3 shows a schematic flowchart of a spatial omics sequencing method according to an embodiment of the present disclosure
- Fig. 4 is the coding sequence structure diagram of cross-localization identification in spatial transcriptome sequencing
- Fig. 5 is the coding sequence structure diagram of the cross-localization marker in spatial proteome sequencing
- Fig. 6 is the coding sequence structure diagram of the cross-localization marker in the spatial epigroup sequencing
- Fig. 7 is the coding sequence structure diagram of the cross-localization marker in the spatial epitranscriptome sequencing
- FIG. 8 shows a schematic flowchart of a method for positioning and marking on a glass slide according to an embodiment of the present disclosure
- FIG. 9 shows a schematic flowchart of a single-cell epitranscriptomic sequencing method according to an embodiment of the present disclosure.
- 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, said antibodies Any of IgM, IgG, IgA, IgD, and IgE.
- FIG. 1 shows a schematic structural diagram of the microfluidic chip according to the 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.
- 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 with 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, and at least two or more microfluidic channels 113 that are respectively connected to the sample inlet holes 111 are connected to the same sample outlet hole 112, and the number of microfluidic channels 113 connected to the sample inlet holes 111 is increased on the chip of the same area.
- the number of injection holes 111 and microfluidic channels 113 is determined.
- the first chip 11 is provided with a sample marking area a, which has microfluidic channels 113 arranged in parallel.
- the spatial sequencing method provided by the embodiment of the present disclosure can be independently completed by using the first chip 11 .
- the sample labeling area a of the first chip 11 covers the sample slice, and the microfluidic channel 113 is in communication with the sample slice.
- the sample holes 112 can be sucked out to make them flow smoothly to the sample marking area, and react with the sample slices in this area, so as to add barcodes on the cell substances of the sample slices.
- the second chip 12 can be bonded with the first chip 11 to be used as a single chip, or can be set as a separate body. When in use, the second chip 12 is superimposed on the first chip 11 for use.
- the material of the first chip 11 is usually polydimethylsiloxane, which is soft and easy to be adsorbed by the pipette tip when adding samples. Methyl methacrylate, the two are used together, and the second chip 12 plays a protective role to avoid direct contact between the pipette tip and the first chip 11 during sample addition, so as to effectively avoid the first chip 11 and the sample slicing due to the adsorption of the pipette tip.
- the slide where it is located is detached to prevent the mixing of independent reagents added to different injection holes 111 of the first chip 11 and cause experiment failure. It can also cooperate with a gun discharger or an automatic pipetting workstation to realize the automation of sample addition.
- 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 also provided with a backflow prevention hole 122 , and the position of the backflow prevention hole 122 is close to the sampling hole 112 .
- a hard object can be used to pass through the backflow prevention hole 122 . , squeeze the region b near the sample outlet hole on the first chip 11 , so that the microfluidic channel 113 in this region b deforms and completely blocks the channel, preventing the backflow of substances from the sample outlet hole 112 . After the reaction is completed, the hard object is taken out, and the squeezed microfluidic channel 113 can recover by itself.
- the third chip 13 is used in conjunction with the first chip 11. Considering that after the number of injection holes 111 and microfluidic channels 113 is increased on the first chip 11, the distance between adjacent microfluidic channels 113 in the sample marking area, the microfluidic The width of the channel 113 can be set to be smaller, which may cause blockage of the microfluidic channel 113 while improving the detection throughput of the sample slice.
- a third chip 13 is added to be superimposed on the first chip 11 or the second chip 12 for use. The substance added in the sample hole 111 flows smoothly to the sample marking area.
- the third chip 13 is provided with a second through hole, and the position of the second through hole corresponds to the position of the sampling hole 112 on the first chip 11 and the position of the backflow prevention hole 122 on the second chip 12 .
- the third chip 13 is also provided with a hollow area corresponding to the position of the sample injection hole 111 , which is superimposed on the first chip 11 or the second chip 12 to form a closed space. It is used to inflate the airtight space, thereby forming a positive pressure above the injection hole 111 .
- the pitch of the microfluidic channel is 100 nm-200 ⁇ m, and/or the width of the microfluidic channel is 100 nm-200 ⁇ m.
- the pitch of the microfluidic channel is 10 ⁇ m
- the width of the microfluidic channel is 20 ⁇ m, which can realize 384 injection holes 113 on the chip area of 11cm x 11cm, so as to realize a total of 147456 kinds of marking combinations of 384 x 384.
- the effective marking rate of slices has also increased to about 44.4%, and the coverage area of sample slices has also reached a coverage of 11.5mm x 11.5mm.
- the number of the injection holes is 10-40,000.
- a total of 589,824 combinations of marking 768 x 768 can be realized, and the sample area of 23.04mm x 23.04mm can be covered under the precision of the microfluidic channel width of 20 ⁇ m and the microfluidic channel width spacing of 10 ⁇ m.
- FIG. 3 shows a schematic flowchart of a spatial omics sequencing method according to an embodiment of the present disclosure. As shown in FIG. 3 , the spatial omics sequencing method includes steps S110-S130.
- step S110 preprocessing sample slices
- step S120 a microfluidic chip is used to add a cross-positioning mark to the sample slice, wherein the cross-positioning mark is determined by adding substances including different barcodes to the injection hole of the microfluidic chip;
- step S130 after cell lysis, amplification and library building are performed on the sample slices in sequence, gene sequencing is performed according to the cross-localization identifier.
- the spatial omics sequencing method uses a microfluidic chip to add substances including different barcodes that mark the position of the cells, the operation steps are simple, and are not limited to spatial transcriptome sequencing, spatial proteome sequencing, spatial epigenetic sequencing, and spatial epitranscriptome sequencing, etc., Wide range of applications.
- the number of sample outlet holes is less than the number of sample inlet holes, which changes the previous design of a microfluidic channel connecting the sample inlet hole and the sample outlet hole, so that more than two sample holes are connected to the sample hole separately.
- the connected microfluidic channels are connected to the same sample outlet, which increases the number of sample injection holes and microfluidic channels.
- the number of microfluidic channels that can form cross-positioning marks increases, and the spacing can be set smaller, thereby improving the spatial omics.
- the detection throughput of the sequencing method and the labeled area of the sample is less than the number of sample inlet holes, which changes the previous design of a microfluidic channel connecting the sample inlet hole and the sample outlet hole, so that more than two sample holes are connected to the sample hole separately.
- the connected microfluidic channels are connected to the same sample outlet, which increases the number of sample injection holes and microfluidic channels.
- the number of microfluidic channels that can form cross-positioning marks increases, and the spacing can be set smaller
- the sample slice may be embryonic tissue, tumor tissue, etc., which is not limited in the present disclosure.
- the preprocessing of the sample slice in step S110 includes at least one of the following manners:
- Preparation of sample slices Preparation of sample slices, staining of sample slices, fixation of sample slices, BSA blocking of sample slices, permeabilization of sample slices, placing sample slices in the sample area in the middle of the glass slide.
- the cross positioning marks are determined by different barcodes.
- the principle is: the sample marking area of the microfluidic chip (the area has parallel microfluidic channels) covers the sample slice, and the first group of barcodes A pass through the microfluidic chip. channels, resulting in parallel and spatially separated rows, each row including A1-AN labels, N being a positive integer greater than 1; the microfluidic chip and sample are subsequently washed, and the microfluidic chip is rotated so that it is perpendicular to the first labeling A second set of barcode B is passed through the microfluidic channel, resulting in parallel and spatially separated columns, each column including B1-BN labels, and N being a positive integer greater than 1.
- each area of the tissue includes a unique composite barcode AiBj(i, j ⁇ N), so as to mark and distinguish different spatial areas.
- step S120 using a microfluidic chip to add a cross-positioning mark to the sample slice includes:
- Second substances containing different barcode B are respectively added to the sample injection holes of the rotated microfluidic chip, and a second reaction is carried out with the sample slices.
- the microfluidic chip used for the second reaction operation may be the same chip as the microfluidic chip used for the first reaction, and it is only necessary to rotate the chip after cleaning the chip after the first reaction.
- the second reaction is sufficient. It can be understood that in order to avoid the influence of the residual liquid in the microfluidic channel after the first reaction on the second reaction, and to avoid affecting the labeling effect of the sample slice, another new microfluidic chip can also be used to operate the second reaction. No restrictions.
- spatial omics sequencing methods specifically spatial transcriptome sequencing, spatial proteome sequencing, spatial epigenetic sequencing, and spatial epitranscriptome sequencing, are described below as examples.
- Fig. 4 is the coding sequence structure diagram of the cross-localization identification in spatial transcriptome sequencing, 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, multi-T sequence, The poly-T sequence is complementary to the poly-A sequence of the mRNA, and the sequence from the 5' end to the 3' end of the second substance comprising barcode B is: amplification sequence, barcode B sequence, unique molecular identifier sequence (UMI sequence) , the linker B sequence, the linker A sequence is complementary to the linker B sequence, or the linker A sequence and the linker B sequence are complementary to each other through the additional linker sequence, after labeling, each region of the tissue includes a unique composite barcode AiBj, In this way, different spatial regions are marked and distinguished.
- UMI sequence unique molecular identifier sequence
- the first reaction performed by adding a first substance comprising barcode A is a reversal reaction
- the second reaction performed by adding a second substance comprising barcode B is a ligation reaction.
- reagents such as RNAbarcode A and reverse enzyme are added
- reagents such as RNAbarcode B ligase are added, with specific reference to the prior art, which will not be repeated in this disclosure.
- Figure 5 is a structural diagram of the coding sequence of the cross-localization marker in spatial proteome sequencing, which is the same as the coding sequence of the cross-localization marker in spatial transcriptome sequencing, and is also composed of a first substance containing barcode A and a second substance containing barcode B. It will not be repeated here.
- a partner coupled to barcode C which includes the barcode from the 5' end to the 3' end, is added to the sample slice and incubated to allow the partner to bind to the protein of interest on the sample slice.
- the C sequence, the poly-A sequence, the barcode C-sequence coupling partner and the poly-A sequence, the poly-A sequence is complementary to the poly-T sequence in the first substance.
- the antibody conjugated with barcode C can be introduced into the sample slice through the microfluidic channel and incubated with the sample slice, or the partner conjugated with barcode C can be added to the sample slice through a pipette, an automatic sample loading workstation, etc. After pre-incubating the sample slice with the tissue, the microfluidic chip is then covered on the sample slice to carry out the first and second reactions.
- Figure 6 is a structural diagram of the coding sequence of the cross-localization marker in the spatial epigenetic group sequencing.
- the partner of the target protein and the corresponding buffer are added to the sample slice, so that the partner can bind to the target protein.
- the first reaction and the second reaction are incubation reactions, and barcode A and barcode B are not connected.
- the first substance added is a reagent such as a pre-embedded PAT mixture with barcode A connectors and corresponding buffers
- the added second substance is pre-embedded with barcode B connectors.
- Reagents such as PAT mixture and corresponding buffer.
- the PAT mixture is a fusion protein formed by Protein A and the transposase Tn5, which has specific antibody targeting and efficient DNA cleavage and linker addition activities.
- Protein A can specifically recognize and bind to the Fc segment of the antibody , and Tn5 can efficiently cut DNA and add linker sequences. Therefore, by incubating the genomic DNA of the tissue with PAT, the target DNA fragment with a specific linker can be obtained.
- PAT interrupts the chromatin of the antibody-binding region to obtain the target DNA fragment.
- an appropriate concentration of EDTA or other reagents that can chelate metal ions such as EGTA is added to terminate the PAT reaction.
- the adapter sequence with barcode A or B in PAT is connected to the target DNA fragment, so that barcode A and barcode B are added at both ends of the DNA fragment.
- the spatial omics sequencing method of the embodiment of the present disclosure is applied to spatial epigenetic sequencing, and high-throughput spatial epigenetic sequencing is realized by adding barcode A and barcode B to both ends of the DNA fragment.
- Tn5-barcode A also includes a partner binding substance, and the partner binding substance is used to bind the target protein partner;
- the chip After removing the microfluidic chip and washing the tissue, the chip was rotated 90 degrees and then covered on the tissue again.
- the mixed solution of pre-embedded Tn5-barcode B and the reaction buffer were added to the injection hole and aspirated out of the sample hole until The microfluidic channel is filled with liquid and incubated for an appropriate time;
- the Tn5-barcode B also includes a partner binding substance, and the partner binding substance is used to bind the target protein partner;
- tissue was dissociated with lysate for subsequent cDNA purification and amplification and library construction and sequencing.
- the Tn5-barcode A containing the partner-binding substance is a fusion protein formed by the partner-binding substance and the transposase Tn5 pre-embedded barcode A linker;
- the Tn5-barcode A containing the partner-binding substance B is the pre-embedded barcode B linker of the fusion protein formed by the partner binding substance and the transposase Tn5;
- the partner is selected from antibody, antibody Fc fragment; the partner binding substance is selected from protein A, protein G, Fc receptor protein.
- Figure 7 is a structural diagram of the coding sequence of the cross-localization mark in spatial transcriptome sequencing, which is the same as the coding sequence of the cross-localization mark in spatial transcriptome sequencing, and also consists of barcode A and barcode B, which will not be repeated here. It should be noted that, when preprocessing sample slices, it is necessary to reverse the RNA with oligo dT or random primers to obtain cDNA, and then add the antibody of the target protein and the corresponding buffer to the sample slice to bind the antibody to the target protein. Specific reference is made to the technical content of the spatial appearance group test, which will not be repeated here.
- DNase I deoxyribonuclease I
- the spatial omics sequencing method of the embodiment of the present disclosure is applied to the spatial epitranscriptome sequencing, and high-throughput spatial epitranscriptome sequencing is realized by adding barcode A and barcode B to both ends of the cDNA fragment.
- Tn5-barcode A also includes a partner binding substance, and the partner binding substance is used to bind the target protein partner;
- the Tn5-barcode B After removing the microfluidic chip and washing the tissue, rotate the chip 90 degrees and cover the tissue again, add the pre-embedded Tn5-barcode B mixed solution and reaction buffer into the injection hole, and aspirate until the microfluidic The channel is filled with liquid and incubated for an appropriate time; the Tn5-barcode B also includes a partner binding substance, and the partner binding substance is used to bind the target protein partner;
- tissue was dissociated with lysate for subsequent cDNA purification and amplification and library construction and sequencing.
- the Tn5-barcode A containing the partner-binding substance is a fusion protein formed by the partner-binding substance and the transposase Tn5 pre-embedded barcode A linker;
- the Tn5-barcode A containing the partner-binding substance B is the pre-embedded barcode B linker of the fusion protein formed by the partner binding substance and the transposase Tn5;
- the partner is selected from antibody, antibody Fc fragment; the partner binding substance is selected from protein A, protein G, Fc receptor protein.
- the spatial omics sequencing method further includes steps S140-S150.
- step S140 in the process of performing the first reaction and/or the second reaction, the injection hole of the microfluidic chip is inflated with positive pressure and/or the microfluidic chip is sucked by negative pressure
- the method of the sample outlet hole enables the first substance and/or the second substance to smoothly enter the sample marking area; wherein, the sample marking area is the area where the microfluidic chip covers the sample slice, and this area has parallel the provided microfluidic channel;
- the method of inflating the sample hole with positive pressure can be combined with the method of inflating the sample hole with negative pressure, so that the first substance and/or the second substance can be added smoothly. Enter the sample marking area.
- step S150 the microfluidic channel is squeezed by a hard object through the anti-reflux hole near the sample outlet, so as to prevent the first material and the /or the second substance flows back from the sample outlet hole of the microfluidic chip.
- the microfluidic channel is squeezed to deform and completely block the channel, so as to prevent the backflow of the material flowing out of the sample outlet, and at the same time, the first material and/or the second material and the cell material of the sample slice can be sufficiently Carry out the reaction, and after the reaction is completed, take out the hard object, and the squeezed microfluidic channel can recover by itself.
- Embodiment 1 The spatial transcriptome sequencing method includes the following steps:
- Fixation of slices Take 7um neonatal mouse brain tissue slices fresh or stored at -80°C, first wash with 300ul 1xPBS (phosphate buffered saline) for 10min, and then use 300ul 4% formaldehyde (1xPBS configuration) to fix 20min;
- 1xPBS phosphate buffered saline
- BSA blocking of sections (optional): Add 300ul 1% BSA (1xPBS+1%RNase Inhibitor (RI) configuration) to the tissue section, incubate for 30min at room temperature, and wash with 300ul1xPBS for 3min.
- RI RNase Inhibitor
- Permeabilization of slices add 300ul of 0.5% TritonX-100 (1xPBS+1%RI configuration) to the tissue slices, incubate at room temperature for 10min, wash with 300ul of 1xPBS for 10min, and finally let the tissue slices dry at room temperature After drying, the tissue sections were attached to the microfluidic chip and fixed with clamps.
- Reverse transcription of tissue RNA Add 5ul of the inversion solution containing specific RNAbarcode A to each injection well and slowly aspirate out the sample well to fill the microfluidic channel with the liquid.
- the inversion solution contains: 1x Maxima H Minus RT buufer, 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 RNAbarcode A. Then, it was placed at room temperature for 30 minutes, then at 42°C for 90 minutes. Finally, the inversion solution was slowly drained and 8ul of 1x Neb buffer 3.1+0.5% RI was added to each injection hole, and the solution was slowly pumped and washed for 10 minutes.
- RNA barcode B connection of tissue RNA: Rotate the microfluidic chip by 90 degrees and cover it on the tissue section again. Add 5ul of ligase solution containing specific RNAbarcode B to each injection well and slowly aspirate out the sample wells to fill the microfluidic channel with the liquid.
- the ligase solution contains: 1x T4 DNA ligase buffer, 0.3U/ul RNase Inhibitor, 0.1U/ul SuperaseIn RNase Inhibito, 0.1% TritonX-100, RNase Free H2O, 0.5x Neb buffer 3.1, 16U/ul T4 DNA ligase and 6uM RNA barcode B.
- Tissue lysis Add 200ul lysis solution to the tissue section, wherein the lysis solution contains: 10mM Tris (pH8.0), 200mM NaCl, 50mM EDTA (pH8.0), 2.2% SDS, Water and 1xPBS. After repeated pipetting, the lysate was recovered into a 1.5ml EP tube, placed in a 55°C metal bath at 600 rpm for lysis for 2h, then taken out and stored at -80°C.
- Template Switch binding to sample cDNA First, wash the magnetic beads bound to sample cDNA. Add a total of 110ul of the prepared template conversion solution to the C1 magnetic beads that bind the sample cDNA and mix.
- the template conversion solution contains: 1x Maxima H Minus RT buufer, 1mM dNTP, 1U/ul RNase Inhibitor, RNase Free H2O, 5U/ul Maxima H Reverse Transcriptase and 2.5uM Template Switch Oligo(TSO). The cells were then incubated at room temperature for 30 min with rotation, and finally incubated with rotation at 42°C for 1 h.
- Amplification of sample cDNA Add 120ul of cDNA amplification buffer to the C1 magnetic beads after template conversion, wherein the cDNA amplification buffer contains: 1x Kapa Hifi Master mix, 0.4uM upstream amplification primer, 0.4uM downstream Amplification primers and Water. After mixing, it was divided into two tubes and placed in a PCR machine for amplification.
- Fragment screening of amplified DNA products Add 84ul Kapa Pure Beads to 120ul amplified product, mix well, and let stand for 5 minutes to bind DNA. Then placed on a magnetic stand, washed twice with 200ul 85% ethanol, dried at room temperature for 3 min, and finally eluted the DNA from the magnetic beads with 20ul RNase Free Water.
- Embodiment 2 The spatial proteome sequencing method includes the following steps:
- Fixation of slices Take 7um neonatal mouse brain tissue slices fresh or stored at -80°C, first wash with 300ul 1xPBS for 10min, and then use 300ul 4% formaldehyde (1xPBS configuration) to fix for 20min;
- BSA blocking of sections (optional): Add 300ul 1% BSA (1xPBS+1%RNase Inhibitor (RI) configuration) to the tissue section, incubate for 30min at room temperature, and wash with 300ul1xPBS for 3min.
- RI RNase Inhibitor
- Incubation of antibodies Add 0.1ug of antibodies coupled with different barcodes to the tissue sections, and incubate at 4°C for 30min to fully bind the antibodies to the protein of interest. Then use the wash buffer of 1%BSA+0.01%Tween 20 in 1x PBS to wash 3 times and RNase Free Water once. After the tissue sections are dried at room temperature, attach the tissue sections to the microfluidic chip, and use a clamp. fix it;
- Reverse transcription of antibody barcode C Add 5ul of inversion solution containing specific RNA barcode A to each injection well and slowly aspirate out the sample well so that the liquid fills the microfluidic channel. Contains: 1x Maxima H Minus RT buufer, 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, it was placed at room temperature for 30 minutes, then at 42°C for 90 minutes. Finally, the inversion solution was slowly drained and 8ul of 1x Neb buffer 3.1+0.5% RI was added to each injection hole, and the solution was slowly pumped and washed for 10 minutes.
- connection of antibody barcode C Rotate the microfluidic chip by 90 degrees and cover it on the tissue section again. Add 5ul of the ligation solution containing specific RNA barcode B to each injection well and slowly aspirate out the wells to fill the microfluidic channel, where the ligase solution contains: 1x T4 DNA ligase buffer, 0.3U/ul RNase Inhibitor, 0.1U/ul SuperaseIn RNase Inhibito, 0.1% TritonX-100, RNase Free H2O, 0.5x Neb buffer 3.1, 16U/ul T4 DNA ligase and 6uM RNA barcode B.
- Tissue lysis Add 200ul lysis solution to the tissue section, wherein the lysis solution contains: 10mM Tris (pH8.0), 200mM NaCl, 50mM EDTA (pH8.0), 2.2% SDS, Water and 1xPBS. After repeated pipetting, the lysate was recovered into a 1.5ml EP tube, placed in a 55°C metal bath at 600 rpm for lysis for 2h, then taken out and stored at -80°C.
- Template Switch binding to antibody barcode C First, wash the magnetic beads bound to antibody barcode C. Add a total of 110ul of the prepared template conversion solution to the C1 magnetic beads bound to antibody barcode C and mix well.
- the template conversion solution contains: 1x Maxima H Minus RT buufer, 1mM dNTP, 1U/ul RNase Inhibitor, RNase Free H2O, 5U/ul ul Maxima H Reverse Transcriptase and 2.5uM Template Switch Oligo(TSO). The cells were then incubated at room temperature for 30 min with rotation, and finally incubated with rotation at 42°C for 1 h.
- Amplification of antibody barcode C Add 120ul of amplification buffer to the C1 magnetic beads after template conversion, wherein the amplification buffer contains: 1x Kapa Hifi Master mix, 0.4uM upstream amplification primer, 0.4uM downstream amplification Amplifier and Water. After mixing, it was divided into two tubes and placed in a PCR machine for amplification.
- Fragment screening of amplified DNA products Add 84ul Kapa Pure Beads to 120ul amplified product, mix well, and let stand for 5 minutes to bind DNA. Then put it on a magnetic stand, aspirate the supernatant into a new EP tube, add 108ul Kapa Pure Beads, and let stand for 5 minutes to bind DNA. Then place it on a magnetic rack, discard the supernatant, wash twice with 200ul 85% ethanol, dry at room temperature for 3 min, and finally use 20ul RNase Free Water to elute the DNA from the magnetic beads.
- FIG. 8 shows a schematic flowchart of a method for positioning and marking on a glass slide according to an embodiment of the present disclosure.
- the spatial omics sequencing method includes steps S210-S240.
- step S210 preprocessing the glass slide
- step S210 add a first substance containing barcode A to the injection hole of the microfluidic chip, and perform a first reaction with the glass slide;
- step S230 the microfluidic chip is rotated at a predetermined angle, so that the direction of the microfluidic channel of the microfluidic chip through which the first substance flows and the direction of the rotated microfluidic channel form a predetermined angle.
- step S240 a second substance containing barcode B is added to the injection hole of the rotated microfluidic chip, and a second reaction is performed with the first substance that has undergone the first reaction on the glass slide; wherein, The barcode A and barcode B are used to mark the cross positioning marks formed on the glass slide.
- a chip with multiple barcode combinations is obtained by connecting barcode A and barcode B on the glass slide.
- the fabricated chip can be placed at -80°C for standby use. It is used in the sequencing of spatial transcriptome, spatial proteome, spatial epigenome, and spatial epitranscriptome.
- the preprocessing of the glass slide includes: coating a sample area of the glass slide with one of gold nanoparticles, streptavidin or amino modification, so that the modified area covers sample area.
- the first substance may be a mixture containing RNA barcode A and a nucleic acid coupling agent, so as to couple the RNA barcode A and the glass slide, wherein, if the gold nanoparticles are modified on the slide glass , the 5' end of RNA barcode A can be modified with sulfhydryl group, if the slide is amino modified, the 5' end of RNA barcode A can be modified with carboxyl group; if streptavidin is modified on the slide, then RNA barcode A The 5' end can be modified with biotin.
- the second substance can be a mixture comprising RNA barcode B and ligase etc. that are pre-annealed with the linker linker sequence, so that the RNA barcode B and the RNA barcode A are linked with the help of the linker linker sequence and the ligase.
- the barcode can be stably fixed on the glass slide, which avoids the labeling loss caused by the barcode being washed away by the washing solution during the washing process; more barcodes are marked on the glass slide through the interaction of streptomycin and biotin. On glass slides, labeling throughput is improved.
- the method for positioning and marking on a glass slide further includes steps S250-S260.
- step S250 in the process of performing the first reaction and/or the second reaction, the first substance and/or the first substance and/or the third The two substances enter the glass slide marking area smoothly; wherein, the glass slide marking area is the central area where the microfluidic chip covers the glass slide, and the area has microfluidic channels arranged in parallel;
- the method of inflating the sample hole with positive pressure can be combined with the method of inflating the sample hole with negative pressure, so that the first substance and/or the second substance can be added smoothly. Enter the sample marking area.
- step S260 the microfluidic channel is squeezed by a hard object through the anti-reflux hole near the sample outlet, so as to prevent the first material and the /or the second substance flows back from the sample outlet hole of the microfluidic chip.
- the microfluidic channel is squeezed to deform and completely block the channel, so as to prevent the backflow of the material flowing out of the sample outlet, and at the same time, the first material and/or the second material and the cell material of the sample slice can be sufficiently Carry out the reaction, and after the reaction is completed, take out the hard object, and the squeezed microfluidic channel can recover by itself.
- barcode A modified with thiol, biotin or carboxyl group to the injection hole, and make it pass through nano-gold (thiol modification), streptavidin (biotin modification) or Amino modification (carboxyl modification) coupled to the glass slide;
- FIG. 9 shows a schematic flowchart of a single-cell epitranscriptomic sequencing method according to an embodiment of the present disclosure. As shown in FIG. 9 , the epitranscriptomic sequencing method includes steps S310-S330.
- step S310 the sample is pretreated
- step S320 a partner of the target protein is added to the pretreated sample, so that the partner of the target protein is combined with the target protein;
- step S330 the sample is incubated with a first substance comprising barcode A and a second substance comprising barcode B, the first substance and the second substance also include Tn5 and a target protein partner binding substance, so The target protein partner binding substance is used to bind the target protein partner, the Tn5 interrupts the cDNA of the sample to form a cDNA fragment, and barcode A and barcode B are added to the cDNA fragment, and then cell lysis is performed in sequence , cDNA amplification and library sequencing.
- the epitranscriptomic sequencing provided in the embodiments of the present disclosure can be applied to single-cell sequencing, and can also be applied to spatial epitranscriptomic sequencing in combination with a microfluidic chip.
- By adding barcode A and barcode B to both ends of the cDNA fragment enabling single-cell epitranscriptomic detection of tissue samples.
- the preprocessing of the sample in step S310 includes:
- Tn5-barcode A and Tn5-barcode B also include a partner binding substance, and the partner binding substance for binding the target protein partner;
- the Tn5 interrupts the chromatin of the sample to form DNA fragments.
- an appropriate concentration of EDTA or other reagents that can chelate metal ions such as EGTA is added to terminate the reaction, and barcode A and barcode are added to the DNA fragment at the same time.
- B and then sequentially use the lysate to lyse the cells, amplify the cDNA, and construct the library for sequencing.
- the Tn5-barcode A containing the partner-binding substance is a fusion protein formed by the partner-binding substance and the transposase Tn5 pre-embedded barcode A linker;
- the Tn5-barcode A containing the partner-binding substance B is the pre-embedded barcode B linker of the fusion protein formed by the partner binding substance and the transposase Tn5;
- the partner is selected from antibody, antibody Fc fragment; the partner binding substance is selected from protein A, protein G, Fc receptor protein.
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Abstract
提供了一种空间组学测序、单细胞表观转录组学测序及定位标识方法,所述方法包括:预处理样品切片;利用微流控芯片将交叉定位标识添加至所述样品切片,其中所述交叉定位标识为向微流控芯片的进样孔加入的包括不同barcode的物质所确定的;依序对所述样品切片进行细胞裂解、扩增及建库后,根据所述交叉定位标识进行基因测序。
Description
相关申请的交叉引用
无。
本公开涉及基因测序技术领域,具体涉及一种空间组学测序、单细胞表观转录组学测序及定位标识方法。
空间异质性是器官功能的关键特征,细胞的位置信息对细胞调控机制和细胞谱系发生过程的研究十分重要。传统的基因测序在组织样本或细胞群的整体上进行,细胞之间的差异可能因平均化被掩盖,单细胞测试技术可以在单细胞层面揭示每个细胞的基因信息,使不同的细胞类型得以区分。但是仍然缺乏揭示细胞在组织体内原有位置信息的能力。因此,需要一种保留细胞原有空间位置信息的测序方法。
发明内容
为了解决相关技术中的问题,本公开实施例提供一种空间组学测序、单细胞表观转录组学测序及定位标识方法。
第一方面,本公开实施例中提供了一种空间组学测序方法。
具体地,所述空间组学测序方法,包括:
预处理样品切片;
利用微流控芯片将交叉定位标识添加至所述样品切片,其中,所述交叉定位标识为向微流控芯片的进样孔加入的包括不同barcode的物质所确定的;
依序对所述样品切片进行细胞裂解、扩增及建库后,根据所述交叉定位标识进行基因测序。
可选地,所述利用微流控芯片将交叉定位标识添加至所述样品切片,包括:
将所述微流控芯片覆盖在样品切片上;
向所述微流控芯片的进样孔分别加入包含不同barcode A的第一物质,与所述样品切片进行第一反应;
使用缓冲液对所述样品切片进行洗涤;
以预定角度旋转所述微流控芯片,使得所述第一物质流经的所述微流控芯片的微流体通道的方向与旋转后的所述微流体通道的方向形成所述交叉定位标识;
向旋转后的所述微流控芯片的进样孔分别加入包含不同barcode B的第二物质,与所述样品切片进行第二反应。
可选地,所述方法还包括:
在进行所述第一反应和/或第二反应的过程中,利用正压充气所述微流控芯片的进样孔和/或负压抽吸所述微流控芯片的出样孔的方式,促使所述第一物质和/或第二物质顺利进入样品标记区域;其中,所述样品标记区域为所述微流控芯片覆盖所述样品切片的区域,该区域具有平 行设置的所述微流体通道;和/或利用硬物穿过所述出样孔附近的防回流孔挤压所述微流体通道,防止所述第一反应和/或第二反应的过程中,所述第一物质和/或第二物质从所述微流控芯片的出样孔回流。
可选地,所述方法应用于空间转录组测序或空间蛋白组测序,其中,所述第一反应为反转反应,第二反应为连接反应,以在所述样品切片的mRNA上添加barcode A和barcode B。
可选地,所述方法应用于空间蛋白组测序,其中,在第一反应前,加入偶联了barcode C的配偶体以结合感兴趣的目标蛋白,之后依次进行所述第一反应、第二反应。
可选地,所述方法应用于空间表观组测序,其中,所述第一物质、第二物质还包括Tn5,所述Tn5打断配偶体结合区的染色质得到目标DNA片段,同时在该DNA片段上添加barcode A和barcode B。
可选地,所述方法应用于空间表观转录组测序,其中,在第一反应前,加入感兴趣蛋白的配偶体以结合感兴趣的蛋白;任选地,在加入配偶体之前还包括使用引物对RNA进行反转处理得到cDNA的步骤。
可选地,所述方法还包括:
在进行RNA反转处理前,利用消化试剂消化所述样品切片的DNA。
可选地,预处理样品切片包括以下方式中的至少一种:
样品切片的制作、样品切片的染色、样品切片的固定、样品切片的BSA封闭、样品切片的通透处理、将样品切片放置于载玻片中间的样品区域。
第二方面,本公开实施例中提供了一种载玻片上的定位标识方法。
具体地,所述载玻片上的定位标识方法包括:
对载玻片进行预处理;
向微流控芯片的进样孔加入包含barcode A的第一物质,与所述载玻片进行第一反应;
以预定角度旋转所述微流控芯片,使得所述第一物质流经的所述微流控芯片的微流体通道的方向与旋转后的所述微流体通道的方向形成所述交叉定位标识;
向旋转后的所述微流控芯片的进样孔加入包含barcode B的第二物质,与所述载玻片上进行过第一反应的第一物质进行第二反应;
其中,所述barcode A和barcode B用于标记在所述载玻片上形成的所述交叉定位标识。
可选地,所述对载玻片进行预处理包括:在载玻片中间的样品区域涂上纳米金颗粒或者链霉亲和素或者进行氨基修饰。
可选地,在进行所述第一反应和/或第二反应的过程中,利用负压抽吸所述微流控芯片的出样孔的方式,促使所述第一物质和/或第二物质顺利进入载玻片标记区域;其中,所述载玻片标记区域为所述微流控芯片覆盖所述载玻片的中心区域,该区域具有平行设置的微流体通道;和/或
利用硬物穿过所述出样孔附近的防回流孔挤压所述微流体通道,防止所述第一反应和/或第二反应的过程中,所述第一物质和/或第二物质从所述微流控芯片的出样孔回流。
可选地,所述方法应用于空间转录组测序、空间蛋白组测序、空间表观组测序、空间表观 转录组测序。
第三方面,本公开实施例中提供了一种单细胞表观转录组学测序方法。
具体地,所述单细胞表观转录组学测序方法包括:
对样品进行预处理;
向预处理后的样品中加入目标蛋白的配偶体,使所述目标蛋白的配偶体与所述目标蛋白相结合;
将所述样品与包含barcode A的第一物质和包含barcode B的第二物质进行孵育反应,所述第一物质和第二物质还包括Tn5和目标蛋白配偶体结合物质,所述目标蛋白配偶体结合物质用于结合所述目标蛋白配偶体,所述Tn5打断所述样品的染色质形成DNA片段,同时在该DNA片段上添加barcode A和barcode B,然后依序进行细胞裂解、cDNA扩增及建库测序。
可选地,所述对样品进行预处理包括:
所述样品中细胞的固定;
使用引物对所述样品中的RNA进行反转处理;
以及包括可选的在固定后使用消化试剂对所述样品的DNA进行消化的步骤。
本公开实施例提供的技术方案可以包括以下有益效果:
本公开实施例的空间组学测序方法,在预处理样品切片后,从微流控芯片的进样孔加入包括barcode的物质,用于形成标记细胞空间位置的交叉定位标识,然后将该交叉定位标识添加到样品切片的细胞物质(例如RNA、DNA或蛋白质),依序进行细胞裂解、扩增及建库后,最后进行测序和数据分析。上述测序方法利用微流控芯片加入标记细胞位置的包括不同barcode的物质,操作步骤简单,并且不限于空间转录组测序、空间蛋白组测序、空间表观组测序和空间表观转录组测序等,应用范围广。上述测试方法利用的微流控芯片,出样孔的数量少于进样孔的数量,改变了以往进样孔与出样孔连通一条微流体通道的设计,使得两个以上与进样孔分别连通的微流体通道与同一个出样孔连通,增加了进样孔和微流体通道的数量,能够形成交叉定位标识的微流体通道数量增多,可以进一步降低微流体通道的间距、宽度,从而提高了空间组学测序方法的检测通量以及样品的标记面积。
应当理解的是,以上的一般描述和后文的细节描述仅是示例性和解释性的,并不能限制本公开。
为了更清楚地说明本公开实施例或相关技术中的技术方案,下面将对示例性实施例或相关技术描述中所需要使用的附图作一简单地介绍,显而易见地,下面描述中的附图是本公开的一些示例性实施例,对于本领域普通技术人员来讲,在不付出创造性劳动的前提下,还可以根据这些附图获得其他的附图。
图1示出根据本公开实施例的微流控芯片的结构示意图;
图2a示出根据本公开实施例的第一芯片的结构示意图;
图2b示出根据本公开实施例的第二芯片的结构示意图;
图3示出根据本公开实施例的空间组学测序方法的流程示意图;
图4为空间转录组测序中交叉定位标识的编码序列结构图;
图5为空间蛋白组测序中交叉定位标识的编码序列结构图;
图6为空间表观组测序中交叉定位标识的编码序列结构图;
图7为空间表观转录组测序中交叉定位标识的编码序列结构图;
图8示出根据本公开实施例的载玻片上的定位标识方法的流程示意图;
图9示出根据本公开实施例的单细胞表观转录组学测序方法的流程示意图。
为了使本技术领域的人员更好地理解本公开方案,下面将结合本公开示例性实施例中的附图,对本公开示例性实施例中的技术方案进行清楚、完整地描述。
在本公开的说明书和权利要求书及上述附图中的描述的一些流程中,包含了按照特定顺序出现的多个操作,但是应该清楚了解,这些操作可以不按照其在本文中出现的顺序来执行或并行执行,操作的序号如101、102等,仅仅是用于区分开各个不同的操作,序号本身不代表任何的执行顺序。另外,这些流程可以包括更多或更少的操作,并且这些操作可以按顺序执行或并行执行。需要说明的是,本文中的“第一”、“第二”等描述,是用于区分不同的消息、设备、模块等,不代表先后顺序,也不限定“第一”和“第二”是不同的类型。
下面将结合本公开示例性实施例中的附图,对本公开示例性实施例中的技术方案进行清楚、完整地描述,显然,所描述的示例性实施例仅仅是本公开一部分实施例,而不是全部的实施例。基于本公开中的实施例,本领域技术人员在没有作出创造性劳动前提下所获得的所有其他实施例,都属于本公开保护的范围。
术语配偶体是指与感兴趣的蛋白结合的分子,包括抗体、适体、抗原结合片段,所述抗体包括完整抗体、重组抗体或抗体片段,如抗体Fc片段、Fab片段,scFv,所述抗体为IgM、IgG、IgA、IgD、IgE中的任一种。
实施例一
本公开实施例提供的空间组学测序方法采用了微流控芯片技术,图1示出根据本公开实施例的微流控芯片的结构示意图。图2a示出根据本公开实施例的第一芯片的结构示意图。图2b示出根据本公开实施例的第二芯片的结构示意图。如图1、图2a、图2b所示,所述微流控芯片10包括:第一芯片11、第二芯片12和第三芯片13。所述第一芯片11包括:进样孔111、出样孔112、连通进样孔111和出样孔112的微流体通道113。所述出样孔112的数量小于进样孔111的数量,并将至少两个以上与进样孔111分别连通的微流体通道113与同一个出样孔112连通,在同样面积的芯片上增加了进样孔111和微流体通道113的数量。
第一芯片11上设置有样品标记区域a,该区域具有平行设置的微流体通道113。本公开实施例提供的空间测序方法可以使用第一芯片11独立完成,使用时,第一芯片11的样品标记区域a覆盖样品切片,微流体通道113与样品切片是连通的,向进样孔111加入包括不同barcode(条形码)的物质后,通过抽吸出样孔112的方式可以使其顺利流向样品标记区域,与该区域 内的样品切片进行反应,从而在样品切片的细胞物质上添加barcode。
所述第二芯片12可以与第一芯片11键合在一起作为一个芯片使用,也可以分体设置,使用时,将第二芯片12叠加在第一芯片11上使用。所述第一芯片11的材质通常是聚二甲基硅氧烷,该材质较软容易在加样时被枪头吸附,第二芯片12可以采用相对第一芯片11较硬的材质,例如聚甲基丙烯酸甲酯,二者配合使用,第二芯片12起到保护作用,避免加样时枪头与第一芯片11直接接触,从而能够有效避免因枪头吸附导致第一芯片11与样品切片所在的载玻片脱离,防止第一芯片11不同进样孔111加入的独立试剂发生混和引起实验失败,还可以与排枪或者自动化移液工作站配合,实现加样的自动化。
具体地,所述第二芯片12上开设有第一通孔121,第一通孔121的位置与第一芯片11上的进样孔111、出样孔112的位置相对应。所述第二芯片12上还开设有防回流孔122,所述防回流孔122的位置靠近所述出样孔112,在添加barcode的过程中,可以利用硬物穿过所述防回流孔122,挤压第一芯片11上靠近出样孔附近的区域b,使得该区域b内的微流体通道113变形并完全闭塞通道,防止物质从所述出样孔112回流。反应完成后,取出硬物,被挤压的微流体通道113可自行恢复。
所述第三芯片13与第一芯片11配合使用,考虑到第一芯片11上增加进样孔111与微流体通道113的数量后,样品标记区域内相邻微流体通道113的间距、微流体通道113的宽度均可以设置的更小,在提高样品切片的检测通量的同时,可能会导致微流体通道113的堵塞。为了避免进样孔111加入的物质堵塞微流体通道,通过增设第三芯片13叠加在第一芯片11或第二芯片12上使用,第三芯片13以正压充气进样孔111的方式促使进样孔111加入的物质顺利流向样品标记区域。具体地,所述第三芯片13上开设有第二通孔,第二通孔的位置与第一芯片11上的出样孔112以及第二芯片12上的防回流孔122的位置相对应。所述第三芯片13上还设置有与所述进样孔111所在位置对应的镂空区域,叠加在第一芯片11或第二芯片12上形成密闭空间,该镂空区域上设置有充气孔,用于在密闭空间充气,进而在进样孔111的上方形成正压。
根据本公开的实施例,所述微流体通道的间距为100nm-200μm,和/或所述微流体通道的宽度为100nm-200μm。
例如,微流体通道的间距为10μm,微流体通道的宽度为20μm,能够实现在11cm x 11cm的芯片面积上,设置384个进样孔113,从而实现384 x 384共147456种标记组合,对样品切片的有效标记率也提升至44.4%左右,且样品切片的覆盖面积也达到了11.5mm x 11.5mm的覆盖度。
以上为示意性说明,本公开方式中,所述进样孔的个数为10-40000个。例如采用768孔,从而实现标记768 x 768共589824种组合,在微流体通道宽度20μm,微流体通道宽度间距10μm的精度下,实现覆盖23.04mm x 23.04mm的样品区域。
图3示出根据本公开实施例的空间组学测序方法的流程示意图。如图3所示,所述空间组学测序方法包括步骤S110-S130。
在步骤S110中,预处理样品切片;
在步骤S120中,利用微流控芯片将交叉定位标识添加至所述样品切片,其中,所述交叉定位标识为向微流控芯片的进样孔加入的包括不同barcode的物质所确定的;
在步骤S130中,依序对所述样品切片进行细胞裂解、扩增及建库后,根据所述交叉定位标识进行基因测序。
本公开实施例提供的空间组学测序方法,在预处理样品切片后,从微流控芯片的进样孔加入包括不同barcode的物质,用于形成标记细胞空间位置的交叉定位标识,然后将该交叉定位标识添加到样品切片的细胞物质(例如RNA、DNA或蛋白质),依序进行细胞裂解、扩增及建库后,最后进行测序和数据分析。上述测序方法利用微流控芯片加入标记细胞位置的包括不同barcode的物质,操作步骤简单,并且不限于空间转录组测序、空间蛋白组测序、空间表观组测序和空间表观转录组测序等,应用范围广。上述测试方法利用的微流控芯片,出样孔的数量少于进样孔的数量,改变了以往进样孔与出样孔连通一条微流体通道的设计,使得两个以上与进样孔分别连通的微流体通道与同一个出样孔连通,增加了进样孔和微流体通道的数量,能够形成交叉定位标识的微流体通道数量增多、间距设置也可以更小,从而提高了空间组学测序方法的检测通量以及样品的标记面积。
根据本公开的实施例,所述样品切片可以是胚胎组织、肿瘤组织等,本公开对此不做限制。
根据本公开的实施例,步骤S110中预处理样品切片包括以下方式中的至少一种:
样品切片的制作、样品切片的染色、样品切片的固定、样品切片的BSA封闭、样品切片的通透处理、将样品切片放置于载玻片中间的样品区域。
根据本公开的实施例,所述交叉定位标识由不同barcode确定,其原理是:微流控芯片的样品标记区域(该区域具有平行微流体通道)覆盖样品切片,第一组barcode A通过微流体通道,从而产生彼此平行且空间上分离的行,各行包括A1-AN标签,N为大于1的正整数;随后洗涤微流控芯片和样品,旋转微流控芯片使其垂直于第一次标记的位置放置,在微流体通道中通入第二组barcode B,从而产生彼此平行且空间上分离的列,各列包括B1-BN标签,N为大于1的正整数。经过标记后,组织的每一个区域包括唯一复合条形码AiBj(i,j∈N),以此来对不同的空间区域进行标记区分。
根据本公开的实施例,步骤S120中所述利用微流控芯片将交叉定位标识添加至所述样品切片,包括:
将所述微流控芯片覆盖在样品切片上;
向所述微流控芯片的进样孔分别加入包含不同barcode A的第一物质,与所述样品切片进行第一反应;
使用缓冲液对所述样品切片进行洗涤;
以预定角度旋转所述微流控芯片,使得所述第一物质流经的所述微流控芯片的微流体通道的方向与旋转后的所述微流体通道的方向形成所述交叉定位标识;
向旋转后的所述微流控芯片的进样孔分别加入包含不同barcode B的第二物质,与所述样品切片进行第二反应。
根据本公开的实施例,进行第二反应操作所用的微流控芯片可以与进行第一反应的微流控 芯片为同一芯片,只需在洗涤干净第一反应后的芯片后再旋转该芯片进行第二反应即可。可以理解,为了避免第一反应后微流体通道内残留液体对第二反应的影响,避免影响样品切片的标记效果,也可以采用另一新的微流控芯片操作第二反应,本公开对此不做限制。
下面分别对空间组学测序方法,具体是空间转录组测序、空间蛋白组测序、空间表观组测序以及空间表观转录组测序为例进行说明。
空间转录组测序
图4为空间转录组测序中交叉定位标识的编码序列结构图,其中,包含barcode A的第一物质自5’端至3’端的序列依次为:linker A序列、barcode A序列、多T序列,所述多T序列与mRNA的多A序列互补结合,包含barcode B的第二物质自5’端至3’端的序列依次为:扩增序列、barcode B序列、唯一分子标识符序列(UMI序列),linker B序列,所述linker A序列与linker B序列互补结合,或者所述linker A序列、linker B序列通过额外的linker序列互补结合,经过标记后,组织的每一个区域包括唯一复合条形码AiBj,以此来对不同的空间区域进行标记区分。
添加包含barcode A的第一物质进行的第一反应为反转反应,添加包含barcode B的第二物质进行的第二反应为连接反应。第一反应时加入RNAbarcode A、反转酶等试剂,第二反应时加入RNAbarcode B连接酶等试剂,具体参照现有技术,本公开对此不予赘述。
空间蛋白组测序
图5为空间蛋白组测序中交叉定位标识的编码序列结构图,与空间转录组测序中交叉定位标识的编码序列相同,同样由含有barcode A的第一物质和含有barcode B的第二物质组成,在此不予赘述。
在进行第一反应前,加入偶联了barcode C的配偶体与样品切片孵育,以使配偶体和样品切片上的感兴趣的蛋白结合,所述的barcode C自5’端至3’包括barcode C序列、多A序列,所述barcode C序列偶联配偶体和多A序列,所述多A序列与第一物质中的多T序列互补结合。所述偶联了barcode C的抗体可以通过微流体通道通入到样品切片上与样品切片孵育,也可通过移液器、全自动加样工作站等方式将偶联了barcode C的配偶体加入到样品切片上与组织预先孵育后,再将微流控芯片覆盖在样品切片上进行第一、第二反应。
空间表观组测序
图6为空间表观组测序中交叉定位标识的编码序列结构图。
空间表观组测序中,在预处理样品切片时,向样品切片加入目标蛋白的配偶体和相应的缓冲液,以使配偶体与目标蛋白结合。第一反应以及第二反应均为孵育反应,并未连接barcode A和barcode B。在第一反应中,加入的第一物质是预包埋带barcode A接头的PAT混合液和相应缓冲液等试剂,在第二反应中,加入的第二物质是预包埋带barcode B接头的PAT混合液和相应缓冲液等试剂。其中,PAT混合液是Protein A与转座酶Tn5形成的融合蛋白,具有特异的抗体靶向性以及高效的DNA切割和添加接头的活性,其中,Protein A可以特异性识别并结合抗体的Fc段,,而Tn5可以高效的对DNA进行切割并添加接头序列。因此,组织的基因组DNA通过与PAT孵育,可以得到加有特定接头的DNA目的片段。第二反应一段时间后,PAT打断 抗体结合区的染色质得到目标DNA片段,反应完成后加入适当浓度的EDTA或其他可以螯合金属离子的试剂如EGTA等以终止PAT的反应。PAT中带有barcode A或B的接头序列与目标DNA片段连接,从而实现在该DNA片段两端添加barcode A和barcode B。
本公开实施例的空间组学测序方法,应用于空间表观组测序中,通过在DNA片段的两端添加barcode A和barcode B,实现了高通量的空间表观组测序。
根据本公开的实施例,应用于空间表观组测序时,包括以下步骤:
冰冻或石蜡组织切片(或临近切片)的染色(可选的);
切片的固定(可选的);
若切片进行了固定,则需使用低渗缓冲液进行染色质开放处理;
向切片加入感兴趣蛋白的配偶体和相应缓冲液,使配偶体与组织中相应蛋白质充分结合;
使用洗涤缓冲液对切片进行洗涤;
将微流控芯片覆盖在组织切片上,向进样孔内加入预包埋Tn5-barcode A的混合溶液和反应缓冲液后抽吸出样孔,至微流体通道内充满液体后孵育适当时间;所述Tn5-barcode A还包括配偶体结合物质,所述配偶体结合物质用于结合所述目标蛋白配偶体;
移除微流控芯片并洗涤组织后,将芯片旋转90度后再次覆盖在组织上,向进样孔内加入预包埋Tn5-barcode B的混合溶液和反应缓冲液抽吸出样孔,至微流体通道内充满液体后孵育适当时间;所述Tn5-barcode B还包括配偶体结合物质,所述配偶体结合物质用于结合所述目标蛋白配偶体;
利用Tn5打断所述样品的染色质形成DNA片段;
向进样孔内加入适当浓度的EDTA或其他可以螯合金属离子的试剂如EGTA等后抽吸出样孔,至微流体通道内充满液体后置于相应温度下反应适当时间以终止反应。
使用裂解液将组织进行解离,用于后续cDNA纯化扩增及建库测序。
在一个实施例中,所述含有配偶体结合物质的Tn5-barcode A为配偶体结合物质与转座酶Tn5形成的融合蛋白预包埋barcode A接头;所述含有配偶体结合物质的Tn5-barcode B为配偶体结合物质与转座酶Tn5形成的融合蛋白预包埋barcode B接头;
在一个实施例中,所述配偶体选自抗体、抗体Fc片段;所述配偶体结合物质选自protein A、protein G、Fc受体蛋白。
空间表观转录组测序
图7为空间表观转录组测序中交叉定位标识的编码序列结构图,与空间转录组测序中交叉定位标识的编码序列相同,同样由barcode A和barcode B组成,在此不予赘述。需要说明的是,预处理样品切片时,需对RNA利用oligo dT或随机引物进行反转处理得到cDNA,之后向样品切片加入目标蛋白的抗体和相应的缓冲液,以使抗体与目标蛋白结合,具体参照空间表观组测试的技术内容,在此不予赘述。另外,为了避免样品切片中原始DNA对反转录得到的cDNA的影响,还可以利用DNase I(脱氧核糖核酸酶I)对原始DNA进行消化处理,之后再进行RNA的反转处理。本公开实施例的空间组学测序方法,应用于空间表观转录组测序中,通过在cDNA片段的两端添加barcode A和barcode B,实现了高通量的空间表观转录组测序。
根据本公开的实施例,应用于空间表观转录组测序时,包括以下步骤:
冰冻或石蜡组织切片(或临近切片)的染色(可选的);
切片的固定(可选的);
使用DNase I对基因组DNA进行消化(可选的);
使用oligo dT或随机引物进行对RNA进行反转处理;
向切片加入感兴趣蛋白的配偶体和相应缓冲液,使配偶体与组织中相应蛋白质充分结合;
使用洗涤缓冲液对切片进行洗涤;
将微流控芯片覆盖在组织切片上,向进样孔内加入预包埋Tn5-barcode A的混合溶液和反应缓冲液后抽吸出样孔,至微流体通道内充满液体后孵育适当时间;所述Tn5-barcode A还包括配偶体结合物质,所述配偶体结合物质用于结合所述目标蛋白配偶体;
移除微流控芯片并洗涤组织后,将芯片旋转90度后再次覆盖在组织上,向进样孔内加入预包埋Tn5-barcode B的混合溶液和反应缓冲液后抽吸,至微流体通道内充满液体后孵育适当时间;所述Tn5-barcode B还包括配偶体结合物质,所述配偶体结合物质用于结合所述目标蛋白配偶体;
利用Tn5打断所述样品的染色质形成DNA片段;
向进样孔内加入适当浓度的EDTA或其他可以螯合金属离子的试剂如EGTA等后抽吸,至微流体通道内充满液体后置于相应温度下反应适当时间以终止反应。
使用裂解液将组织进行解离,用于后续cDNA纯化扩增及建库测序。
在一个实施例中,所述含有配偶体结合物质的Tn5-barcode A为配偶体结合物质与转座酶Tn5形成的融合蛋白预包埋barcode A接头;所述含有配偶体结合物质的Tn5-barcode B为配偶体结合物质与转座酶Tn5形成的融合蛋白预包埋barcode B接头;
在一个实施例中,所述配偶体选自抗体、抗体Fc片段;所述配偶体结合物质选自protein A、protein G、Fc受体蛋白。
根据本公开的实施例,所述空间组学测序方法还包括步骤S140-S150。
在步骤S140中,在进行所述第一反应和/或第二反应的过程中,利用正压充气所述微流控芯片的进样孔和/或负压抽吸所述微流控芯片的出样孔的方式,促使所述第一物质和/或第二物质顺利进入样品标记区域;其中,所述样品标记区域为所述微流控芯片覆盖所述样品切片的区域,该区域具有平行设置的所述微流体通道;
在本公开方式中,为了避免微流体通道的堵塞,可以在负压抽吸出样孔的同时,结合正压充气进样孔的方式,使得加入的第一物质和/或第二物质能够顺利进入样品标记区域。
在步骤S150中,利用硬物穿过所述出样孔附近的防回流孔挤压所述微流体通道,防止所述第一反应和/或第二反应的过程中,所述第一物质和/或第二物质从所述微流控芯片的出样孔回流。
在本公开方式中,通过挤压微流体通道使其变形并完全闭塞通道,以防止出样孔流出的物质回流,同时也可以使第一物质和/或第二物质与样品切片的细胞物质充分进行反应,待反应完成后,取出硬物,被挤压的微流体通道可自行恢复。
实施例1:空间转录组测序方法包括如下步骤:
1、切片的固定(可选的):取新鲜或-80℃储存的7um新生小鼠脑组织切片,首先使用300ul1xPBS(磷酸盐缓冲液)洗涤10min,再使用300ul 4%甲醛(1xPBS配置)固定20min;
2、冰冻或石蜡组织切片(或临近切片)的染色(可选的):将组织切片依次置于100%无水乙醇脱水20s,75%乙醇脱水1.5min,然后加入1%甲酚紫染色液染色2min,而后75%乙醇瞬时冲洗,100%无水乙醇瞬时冲洗,而后晾干拍照。
3、切片的BSA封闭(可选的):向组织切片上加上300ul 1%BSA(1xPBS+1%RNase Inhibitor(RI)配置),室温孵育30min后用300ul 1xPBS洗涤3分钟。
4、切片的通透处理(可选的):向组织切片上加上300ul 0.5%TritonX-100(1xPBS+1%RI配置),室温孵育10min后用300ul 1xPBS洗涤10min,最后待组织切片室温晾干后,将组织切片与微流控芯片进行贴合,并用夹具将其固定。
5、组织RNA的反转录:向每个进样孔中单独加入5ul含有特异性RNAbarcode A的反转液并缓慢抽吸出样孔,以使液体充满微流体通道,其中反转液包含:1x Maxima H Minus RT buufer,500uM dNTP,0.3U/ul SuperaseIn RNase Inhibitor,0.3U/ul RNase Inhibitor,0.05xPBS,RNase Free H2O,20U/ul Maxima H Reverse Transcriptase及3uM RNAbarcode A。随后先置于室温反应30min,再置于42℃反应90min,最后将反转液缓慢抽干后向每个进样孔加入8ul 1x Neb buffer 3.1+0.5%RI,缓慢抽吸洗涤10min。
6、组织RNA的连接:将微流控芯片旋转90度后再次覆盖在组织切片上。向每个进样孔中单独加入5ul含有特异性RNAbarcode B的连接液并缓慢抽吸出样孔,以使液体充满微流体通道,其中连接液包含:1x T4 DNA ligase buffer,0.3U/ul RNase Inhibitor,0.1U/ul SuperaseIn RNase Inhibito,0.1%TritonX-100,RNase Free H2O,0.5x Neb buffer 3.1,16U/ul T4 DNA ligase及6uM RNA barcode B。随后先置于室温反应30min,将连接液剩余部分缓慢抽干,而后向每个进样孔加入10ul 0.1%Triton X-100+0.5%RI,缓慢抽吸洗涤10min后移去微流控芯片。
7、组织裂解:向组织切片中加入200ul裂解液,其中裂解液包含:10mM Tris(pH8.0),200mM NaCl,50mM EDTA(pH8.0),2.2%SDS,Water及1xPBS。反复吹打后将裂解液回收至1.5ml EP管中,置于55℃金属浴600rpm裂解2h,随后取出并置于-80℃保存。
8、样品与链霉亲和素磁珠(Myone C1beads)的结合:向200ul样品裂解液中加入100ul洗涤好的C1beads,室温旋转孵育30min。由于RNA barcode B的5’端带有生物素修饰,因此可以使用链霉亲和素的磁珠对样品cDNA进行富集。
9、结合样品cDNA的模板转换(Template Switch):首先对结合样品cDNA的磁珠进行洗涤。将配置好的共110ul模板转换液加入结合样品cDNA的C1磁珠中混匀,其中模板转换液包含:1x Maxima H Minus RT buufer,1mM dNTP,1U/ul RNase Inhibitor,RNase Free H2O,5U/ul Maxima H Reverse Transcriptase及2.5uM Template Switch Oligo(TSO)。随后室温旋转孵育30min,最后42℃旋转孵育1h。
10、样品cDNA的扩增:向模板转换后的C1磁珠中加入120ul的cDNA扩增缓冲液,其中cDNA扩增缓冲液包含:1x Kapa Hifi Master mix,0.4uM上游扩增引物,0.4uM下游扩增引 物及Water。随后混匀后均分成两管并置于PCR仪中进行扩增。
11、扩增DNA产物的片段筛选:向120ul扩增产物中加入84ul Kapa Pure Beads后混匀,静置5分钟以结合DNA。而后置于磁力架,使用200ul 85%乙醇洗涤2次,室温晾干3min,最后使用20ul RNase Free Water从磁珠上将DNA进行洗脱。
实施例2:空间蛋白组测序方法包括如下步骤:
1、切片的固定(可选的):取新鲜或-80℃储存的7um新生小鼠脑组织切片,首先使用300ul1xPBS洗涤10min,再使用300ul 4%甲醛(1xPBS配置)固定20min;
2、冰冻或石蜡组织切片(或临近切片)的染色(可选的):将组织切片依次置于100%无水乙醇脱水20s,75%乙醇脱水1.5min,然后加入1%甲酚紫染色液染色2min,而后75%乙醇瞬时冲洗,100%无水乙醇瞬时冲洗,而后晾干拍照。
3、切片的BSA封闭(可选的):向组织切片上加上300ul 1%BSA(1xPBS+1%RNase Inhibitor(RI)配置),室温孵育30min后用300ul 1xPBS洗涤3分钟。
4、切片的通透处理(可选的):向组织切片上加上300ul 0.5%TritonX-100(1xPBS+1%RI配置),室温孵育10min后用300ul 1xPBS洗涤10min,
5、抗体的孵育:向组织切片中加入0.1ug耦联了不同barcode的抗体,4℃孵育30min,以使抗体和感兴趣蛋白进行充分结合。再使用1x PBS配置的1%BSA+0.01%Tween 20的wash buffer洗3次及RNase Free Water洗1次,待组织切片室温晾干后,将组织切片与微流控芯片进行贴合,并用夹具将其固定;
6、抗体barcode C的反转录:向每个进样孔中单独加入5ul含有特异性RNA barcode A的反转液并缓慢抽吸出样孔,以使液体充满微流体通道,其中反转液包含:1x Maxima H Minus RT buufer,500uM dNTP,0.3U/ul SuperaseIn RNase Inhibitor,0.3U/ul RNase Inhibitor,0.05xPBS,RNase Free H2O,20U/ul Maxima H Reverse Transcriptase及3uM RNA barcode A。随后先置于室温反应30min,再置于42℃反应90min,最后将反转液缓慢抽干后向每个进样孔加入8ul 1x Neb buffer 3.1+0.5%RI,缓慢抽吸洗涤10min。
7、抗体barcode C的连接:将微流控芯片旋转90度后再次覆盖在组织切片上。向每个进样孔中单独加入5ul含有特异性RNA barcode B的连接液并缓慢抽吸出样孔,以使液体充满微流体通道,其中连接液包含:1x T4 DNA ligase buffer,0.3U/ul RNase Inhibitor,0.1U/ul SuperaseIn RNase Inhibito,0.1%TritonX-100,RNase Free H2O,0.5x Neb buffer 3.1,16U/ul T4 DNA ligase及6uM RNA barcode B。随后先置于室温反应30min,将连接液剩余部分缓慢抽干,而后向每个进样孔加入10ul 0.1%Triton X-100+0.5%RI,缓慢抽吸洗涤10min后移去微流控芯片。
8、组织裂解:向组织切片中加入200ul裂解液,其中裂解液包含:10mM Tris(pH8.0),200mM NaCl,50mM EDTA(pH8.0),2.2%SDS,Water及1xPBS。反复吹打后将裂解液回收至1.5ml EP管中,置于55℃金属浴600rpm裂解2h,随后取出并置于-80℃保存。
9、样品与链霉亲和素磁珠(Myone C1 beads)的结合:向200ul样品裂解液中加入100ul洗 涤好的C1beads,室温旋转孵育30min。由于RNA barcode B的5’端带有生物素修饰,因此可以使用链霉亲和素的磁珠对样品cDNA进行富集。
10、结合抗体barcode C的模板转换(Template Switch):首先对结合抗体barcode C的磁珠进行洗涤。将配置好的共110ul模板转换液加入结合抗体barcode C的C1磁珠中混匀,其中模板转换液包含:1x Maxima H Minus RT buufer,1mM dNTP,1U/ul RNase Inhibitor,RNase Free H2O,5U/ul Maxima H Reverse Transcriptase及2.5uM Template Switch Oligo(TSO)。随后室温旋转孵育30min,最后42℃旋转孵育1h。
11、抗体barcode C的扩增:向模板转换后的C1磁珠中加入120ul的扩增缓冲液,其中扩增缓冲液包含:1x Kapa Hifi Master mix,0.4uM上游扩增引物,0.4uM下游扩增引物及Water。随后混匀后均分成两管并置于PCR仪中进行扩增。
12、扩增DNA产物的片段筛选:向120ul扩增产物中加入84ul Kapa Pure Beads后混匀,静置5分钟以结合DNA。而后置于磁力架,将上清吸出至新的EP管,加入108ul Kapa Pure Beads,静置5分钟以结合DNA。而后置于磁力架,弃上清,使用200ul 85%乙醇洗涤2次,室温晾干3min,最后使用20ul RNase Free Water从磁珠上将DNA进行洗脱。
13、将分选洗脱的DNA使用相关建库试剂盒参照商家产品说明进行建库测序。
实施例二
图8示出根据本公开实施例的载玻片上的定位标识方法的流程示意图。如图8所示,所述空间组学测序方法包括步骤S210-S240。
在步骤S210中,对载玻片进行预处理;
在步骤S210中,向微流控芯片的进样孔加入包含barcode A的第一物质,与所述载玻片进行第一反应;
在步骤S230中,以预定角度旋转所述微流控芯片,使得所述第一物质流经的所述微流控芯片的微流体通道的方向与旋转后的所述微流体通道的方向形成所述交叉定位标识;
在步骤S240中,向旋转后的所述微流控芯片的进样孔加入包含barcode B的第二物质,与所述载玻片上进行过第一反应的第一物质进行第二反应;其中,所述barcode A和barcode B用于标记在所述载玻片上形成的所述交叉定位标识。
本公开实施例的载玻片上的定位标识方法,通过将barcode A和barcode B连接在载玻片上,获得一个具有多种barcode组合的芯片,制作好的芯片可以放置于-80℃备用,具体可以运用于空间转录组、空间蛋白组、空间表观组、空间表观转录组等测序方面。
根据本公开的实施例,所述对载玻片进行预处理包括:在载玻片的样品区域涂覆纳米金颗粒、链霉亲和素或氨基修饰中的一种,使所述修饰区域覆盖样品区域。
根据本公开的实施例,第一物质可以是包含RNA barcode A和核酸偶联剂等混合液,以使RNA barcode A和载玻片之间进行偶联,其中,若载玻片上修饰纳米金颗粒,则RNA barcode A的5’端可进行巯基修饰,若载玻片上为氨基修饰,则RNA barcode A的5’端可进行羧基修饰;若载玻片上修饰链霉亲和素,则RNA barcode A的5’端可进行生物素修饰。第二物质可以是包含预先与linker连接序列退火的RNA barcode B和连接酶等的混合液,以使RNA barcode B与RNA barcode A在linker连接序列和连接酶的帮助下进行连接。
通过上述修饰,能够将barcode稳定的固定于载玻片上,避免了洗涤过程中barcode被洗涤液冲走而带来的标记损失;通过链霉素和生物素的相互作用将更多的barcode标记于载玻片上,提高了标记通量。
根据本公开的实施例,所述载玻片上的定位标识方法还包括步骤S250-S260。
在步骤S250中,在进行所述第一反应和/或第二反应的过程中,利用负压抽吸所述微流控芯片的出样孔的方式,促使所述第一物质和/或第二物质顺利进入载玻片标记区域;其中,所述载玻片标记区域为所述微流控芯片覆盖所述载玻片的中心区域,该区域具有平行设置的微流体通道;
在本公开方式中,为了避免微流体通道的堵塞,可以在负压抽吸出样孔的同时,结合正压充气进样孔的方式,使得加入的第一物质和/或第二物质能够顺利进入样品标记区域。
在步骤S260中,利用硬物穿过所述出样孔附近的防回流孔挤压所述微流体通道,防止所述第一反应和/或第二反应的过程中,所述第一物质和/或第二物质从所述微流控芯片的出样孔回流。
在本公开方式中,通过挤压微流体通道使其变形并完全闭塞通道,以防止出样孔流出的物质回流,同时也可以使第一物质和/或第二物质与样品切片的细胞物质充分进行反应,待反应完成后,取出硬物,被挤压的微流体通道可自行恢复。
下面说明载玻片上的定位标识的具体实验流程如下:
1、在载玻片样品区域涂覆纳米金颗粒、链霉亲和素或者对载玻片样品区域进行氨基修饰;
2、将微流控芯片覆盖在载玻片上,向进样孔加入巯基、生物素或羧基修饰的barcode A,使其通过纳米金(巯基修饰)、链霉亲和素(生物素修饰)或者氨基修饰(羧基修饰)偶联到玻片上;
3、洗涤去除多余的未结合的barcode A;
4、将微流控芯片旋转90度后再次覆盖在载玻片上,向进样孔内加入包含barcode B和T4连接酶的混合液,使barcode B与barcode A进行连接;
5、洗涤去除多余的未结合的barcode B;
6、将制作好的barcode芯片置于-80℃,以备使用。
实施例三
图9示出根据本公开实施例的单细胞表观转录组学测序方法的流程示意图。如图9所示,所述表观转录组学测序方法包括步骤S310-S330。
在步骤S310中,对样品进行预处理;
在步骤S320中,向预处理后的样品中加入目标蛋白的配偶体,使所述目标蛋白的配偶体与所述目标蛋白相结合;
在步骤S330中,将所述样品与包含barcode A的第一物质和包含barcode B的第二物质进行孵育反应,所述第一物质和第二物质还包括Tn5和目标蛋白配偶体结合物质,所述目标蛋白配偶体结合物质用于结合所述目标蛋白配偶体,所述Tn5打断所述样品的cDNA形成cDNA片 段,同时在该cDNA片段上添加barcode A和barcode B,然后依序进行细胞裂解、cDNA扩增及建库测序。
本公开实施例提供的表观转录组学测序可以应用于单细胞测序,同时也可以结合微流控芯片应用于空间表观转录组学测序,通过在cDNA片段的两端添加barcode A和barcode B,从而实现了对组织样品的单细胞表观转录组检测。
根据本公开的实施例,步骤S310中所述对样品进行预处理包括:
所述样品中细胞的固定;
使用引物对所述样品中的RNA进行反转处理;
以及包括可选的在固定后使用消化试剂对基因组DNA进行消化的步骤。
根据本公开的实施例,应用于单细胞表观转录组空间组学测序时,包括以下步骤:
将细胞固定于载玻片上;
可选的包括使用消化试剂对基因组DNA进行消化的步骤;
使用引物对所述细胞的RNA进行反转处理;
向预处理后的细胞中加入感兴趣蛋白的配偶体,使所述感兴趣蛋白的配偶体与所述感兴趣蛋白充分结合;
使用洗涤缓冲液对细胞进行洗涤;
向细胞中加入预包埋Tn5-barcode A和Tn5-barcode B的混合溶液和反应缓冲液进行孵育;所述Tn5-barcode A、Tn5-barcode B还包括配偶体结合物质,所述配偶体结合物质用于结合所述目标蛋白配偶体;
所述Tn5打断所述样品的染色质形成DNA片段,反应完成后加入适当浓度的EDTA或其他可以螯合金属离子的试剂如EGTA等以终止反应,同时在该DNA片段上添加barcode A和barcode B,然后依序使用裂解液将细胞裂解、cDNA扩增及建库测序。
在一个实施例中,所述含有配偶体结合物质的Tn5-barcode A为配偶体结合物质与转座酶Tn5形成的融合蛋白预包埋barcode A接头;所述含有配偶体结合物质的Tn5-barcode B为配偶体结合物质与转座酶Tn5形成的融合蛋白预包埋barcode B接头;
在一个实施例中,所述配偶体选自抗体、抗体Fc片段;所述配偶体结合物质选自protein A、protein G、Fc受体蛋白。
以上描述仅为本公开的较佳实施例以及对所运用技术原理的说明。本领域技术人员应当理解,本公开中所涉及的发明范围,并不限于上述技术特征的特定组合而成的技术方案,同时也应涵盖在不脱离所述发明构思的情况下,由上述技术特征或其等同特征进行任意组合而形成的其它技术方案。例如上述特征与本公开中公开的(但不限于)具有类似功能的技术特征进行互相替换而形成的技术方案。
Claims (15)
- 一种空间组学测序方法,其特征在于,包括:预处理样品切片;利用微流控芯片将交叉定位标识添加至所述样品切片,其中,所述交叉定位标识为向微流控芯片的进样孔加入的包括不同barcode的物质所确定的;依序对所述样品切片进行细胞裂解、扩增及建库后,根据所述交叉定位标识进行基因测序。
- 根据权利要求1所述的空间组学测序方法,其特征在于,所述利用微流控芯片将交叉定位标识添加至所述样品切片,包括:将所述微流控芯片覆盖在样品切片上;向所述微流控芯片的进样孔分别加入包含不同barcode A的第一物质,与所述样品切片进行第一反应;使用缓冲液对所述样品切片进行洗涤;以预定角度旋转所述微流控芯片,使得所述第一物质流经的所述微流控芯片的微流体通道的方向与旋转后的所述微流体通道的方向形成所述交叉定位标识;向旋转后的所述微流控芯片的进样孔分别加入包含不同barcode B的第二物质,与所述样品切片进行第二反应。
- 根据权利要求2所述的空间组学测序方法,其特征在于,还包括:在进行所述第一反应和/或第二反应的过程中,利用正压充气所述微流控芯片的进样孔和/或负压抽吸所述微流控芯片的出样孔的方式,促使所述第一物质和/或第二物质顺利进入样品标记区域;其中,所述样品标记区域为所述微流控芯片覆盖所述样品切片的区域,该区域具有平行设置的所述微流体通道;和/或利用硬物穿过所述出样孔附近的防回流孔挤压所述微流体通道,防止所述第一反应和/或第二反应的过程中,所述第一物质和/或第二物质从所述微流控芯片的出样孔回流。
- 根据权利要求2或3所述的空间组学测序方法,其特征在于,应用于空间转录组测序或空间蛋白组测序,其中,所述第一反应为反转反应,第二反应为连接反应,以在所述样品切片的mRNA上添加barcode A和barcode B。
- 根据权利要求4所述的空间组学测序方法,其特征在于,应用于空间蛋白组测序,其中,在第一反应前,加入偶联了barcode C的配偶体以结合感兴趣的目标蛋白,之后依次进行所述第一反应、第二反应。
- 根据权利要求2或3所述的空间组学测序方法,其特征在于,应用于空间表观组测序,其中,所述第一物质、第二物质还包括Tn5,所述Tn5打断配偶体结合区的染色质得到目标DNA片段,同时在该DNA片段上添加barcode A和barcode B。
- 根据权利要求6所述的空间组学测序方法,其特征在于,应用于空间表观转录组测序,其中,在第一反应前,加入感兴趣蛋白的配偶体以结合感兴趣的蛋白;任选地,在加入配偶体之前还包括使用引物对RNA进行反转处理得到cDNA的步骤。
- 根据权利要求7所述的空间组学测序方法,其特征在于,还包括:在进行RNA反转处理前,利用消化试剂消化所述样品切片的DNA。
- 根据权利要求4-8任一项所述的空间组学测序方法,其特征在于,预处理样品切片包括以下方式中的至少一种:样品切片的制作、样品切片的染色、样品切片的固定、样品切片的BSA封闭、样品切片的通透处理、将样品切片放置于载玻片中间的样品区域。
- 一种载玻片上的定位标识方法,其特征在于,包括:对载玻片进行预处理;向微流控芯片的进样孔加入包含barcode A的第一物质,与所述载玻片进行第一反应;以预定角度旋转所述微流控芯片,使得所述第一物质流经的所述微流控芯片的微流体通道的方向与旋转后的所述微流体通道的方向形成所述交叉定位标识;向旋转后的所述微流控芯片的进样孔加入包含barcode B的第二物质,与所述载玻片上进行过第一反应的第一物质进行第二反应;其中,所述barcode A和barcode B用于标记在所述载玻片上形成的所述交叉定位标识。
- 根据权利要求10所述的方法,其特征在于,所述对载玻片进行预处理包括:在载玻片中间的样品区域涂上纳米金颗粒或者链霉亲和素或者进行氨基修饰。
- 根据权利要求10所述的方法,其特征在于:在进行所述第一反应和/或第二反应的过程中,利用负压抽吸所述微流控芯片的出样孔的方式,促使所述第一物质和/或第二物质顺利进入载玻片标记区域;其中,所述载玻片标记区域为所述微流控芯片覆盖所述载玻片的中心区域,该区域具有平行设置的微流体通道;和/或利用硬物穿过所述出样孔附近的防回流孔挤压所述微流体通道,防止所述第一反应和/或第二反应的过程中,所述第一物质和/或第二物质从所述微流控芯片的出样孔回流。
- 根据权利要求10至12任一项所述的方法,其特征在于,所述方法应用于空间转录组测序、空间蛋白组测序、空间表观组测序、空间表观转录组测序。
- 一种单细胞表观转录组学测序方法,其特征在于,包括:对样品进行预处理;向预处理后的样品中加入目标蛋白的配偶体,使所述目标蛋白的配偶体与所述目标蛋白相结合;将所述样品与包含barcode A的第一物质和包含barcode B的第二物质进行孵育反应,所述第一物质和第二物质还包括Tn5和目标蛋白配偶体结合物质,所述目标蛋白配偶体结合物质用于结合所述目标蛋白配偶体,所述Tn5打断所述样品的染色质形成DNA片段,同时在该DNA片段上添加barcode A和barcode B,然后依序进行细胞裂解、cDNA扩增及建库测序。
- 根据权利要求14所述的方法,其特征在于,所述对样品进行预处理包括:所述样品中细胞的固定;使用引物对所述样品中的RNA进行反转处理;以及包括可选的在固定后使用消化试剂对所述样品的DNA进行消化的步骤。
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