Method for detecting accessibility of space chromatin and application thereof

Abstract

The invention discloses a method for detecting accessibility of space chromatin and application of the method. The method comprises the following steps: (1) slicing a tissue sample to be detected on a chip with spatial information; (2) fixing, permeabilizing and dyeing a tissue sample to be detected; (3) preparing fragmented genome DNA (deoxyribonucleic acid); (4) performing DNA hybridization based on a transfer mode; (5) carrying out connection and terminal repair reaction on the fragmented genome DNA by using DNA polymerase and DNA ligase; (6) denaturation and melting; and (7) sequencing. According to the method, the transcriptional activity state of chromatin can be reflected through chromatin accessibility sequencing analysis, staining imaging and chromatin accessibility information can be obtained on the same tissue slice at the same time without complex micro-fluidic equipment, experimental operation is easy, repeatability is high, and the analysis result is more remarkable.

Description

Technical Field

[0001] The present invention belongs to the field of spatial chromatin accessibility (ATAC) omics sequencing technology and relates to a method for detecting spatial chromatin accessibility and its application. Background Art

[0002] Spatial ATAC-seq is a new spatial omics technology based on NGS (next-generation sequencing) that enables the analysis of chromatin openness of cells at the spatial level. Although single-cell sequencing can unbiasedly define cell types and states, it cannot obtain spatial distribution information of biomolecules and cells in tissues. The function of cells in tissues depends on the local environment in which they are located. The emergence of spatial transcriptomics makes up for the shortcomings of single-cell sequencing, allowing us to describe cell function and state in a natural tissue environment. There are few studies related to spatial ATAC omics sequencing technology. Existing known technologies include:

[0003] Solid-phase technology, spatial ATAC, applies transposase treatment to tissue sections to release chromatin and label DNA fragments, and then uses barcoded solid-phase capture technology to capture and sequence these DNA fragments. This method can obtain whole-genome information with spatial resolution, revealing the spatial changes in gene expression regulatory programs, especially their role in organogenesis, cell lineage differentiation and human pathological processes.

[0004] DBiT technology uses a series of microfluidic channels pressed against fixed and Tn5 transposed tissue samples to create a physical barrier. In DBiT-seq (deterministic barcoding in tissues for spatial omics sequencing), a microfluidic chip is first used to introduce a set of spatial barcodes (Ai), which are covalently bound to a universal linker chain through templated ligation. Then, another microfluidic chip with microchannels perpendicular to the flow direction of the first chip is used to introduce another set of spatial barcodes (Bj). These barcodes are connected to the Ai barcodes to form spatial barcode tissue pixels, each defined by a unique combination of Ai and Bj. Finally, the barcoded complementary DNA and genomic DNA fragments are released after reversing the cross-linking. However, it is impossible to perform staining imaging on the same slice, and complex microfluidic equipment is required to add characteristic barcode information after each sequence to determine its spatial information.

[0005] Slide-seq technology: This technology hybridizes / links unique spatial barcode marker sequences on a solid phase, captures DNA from tissues via a bridging primer, and can use different barcoded magnetic beads to capture genomes from different cells for sequencing analysis. However, this technology relies on performing ATAC-seq and H&E staining separately on the same slide, making it impossible to perform simultaneous ATAC-omics analysis on the same slide.

[0006] Although these technologies are all ATAC-omics technologies, these spatial omics measurements are supplemented by histological staining of adjacent tissue sections, such as H&E staining, and then combined with morphological annotation. None of them can perform imaging and ATAC-omics analysis simultaneously on the same section. Most of them require the use of other microfluidics technologies or UV cutting technologies, which are relatively complex to operate and require the use of special instruments to complete the entire experiment. In addition, they are prone to DNA spillover, resulting in low resolution, uneven subsequent sequencing analysis, and poor results.

[0007] Therefore, there is an urgent need to provide a method that can simultaneously perform staining imaging and chromatin accessibility sequencing analysis on the same tissue section. Summary of the Invention

[0008] In response to the deficiencies in the existing technology and actual needs, the present invention provides a method for detecting spatial chromatin accessibility and its application. The method of the present invention can simultaneously perform staining imaging and chromatin accessibility sequencing analysis on the same tissue section. Chromatin accessibility sequencing analysis can reflect the transcriptional activity state of chromatin, which is an important direction for studying gene expression regulation. It plays an important role in epigenetic mapping, cell differentiation and development, and the occurrence and development of various diseases. Without the need for complex microfluidic equipment, staining imaging and chromatin accessibility information can be simultaneously obtained on the same tissue section. The experimental operation is simple, the repeatability is high, and the analysis results are more significant.

[0009] In order to achieve the purpose of the invention, the present invention adopts the following technical solutions:

[0010] In a first aspect, the present invention provides a method for detecting spatial chromatin accessibility, the method comprising the following steps:

[0011] (1) Slice the tissue sample to be tested on a chip with spatial information;

[0012] (2) Fixing, permeabilizing, and staining the tissue samples to be tested;

[0013] (3) mixing the permeabilized and stained tissue sample with the transposon sequence inserted and the transposase to obtain fragmented genomic DNA;

[0014] (4) DNA hybridization based on transfer mode to capture fragmented genomic DNA;

[0015] (5) using DNA polymerase and DNA ligase to perform ligation and end-repair reactions on the fragmented genomic DNA;

[0016] (6) denaturation and melting to obtain fragmented genomic DNA with spatial information;

[0017] (7) Sequencing analysis.

[0018] The method of the present invention can simultaneously perform staining imaging and chromatin accessibility sequencing analysis on the same tissue section. Chromatin accessibility sequencing analysis can reflect the transcriptional activity state of chromatin, which is an important direction for studying gene expression regulation. It plays an important role in epigenetic mapping, cell differentiation and development, and the occurrence and development of various diseases. Without the need for complex microfluidic equipment, staining imaging and chromatin accessibility information can be simultaneously obtained on the same tissue section. The experimental operation is simple, the repeatability is high, and the analysis results are more significant.

[0019] Preferably, the chip with spatial information in step (1) includes a substrate, the substrate has microspheres, and each microsphere has a capture probe; the capture probe includes a sequencing primer fragment, a barcode fragment, a specific molecular tag and a capture structure for capturing fragmented genomic DNA connected in sequence from the substrate upward.

[0020] Preferably, the chip carrying spatial information contains an anti-overflow protective film.

[0021] Preferably, the anti-overflow protective film has a thickness of 10 μm-100 μm, for example, 10 μm, 20 μm, 30 μm, 50 μm, 80 μm or 100 μm.

[0022] Preferably, the capture probes on the same microsphere in the chip with spatial information contain the same spatial barcode.

[0023] Preferably, the capture probe is modified.

[0024] Preferably, the modification includes any one of phosphorylation modification and biotin modification.

[0025] The tissue slices described in the present invention can be selected from plants, animals and / or microorganisms. The tissue sample to be tested can be obtained from a subject (e.g., by surgical biopsy, whole subject sectioning) or grown in vitro as a cell population on a growth substrate or culture dish and prepared as a tissue cross-section or tissue slice for analysis. The grown sample can be thin enough to be analyzed without further processing steps. The thickness of the tissue slice can be a fraction of the maximum cross-sectional dimension of the cell, or a tissue slice with a thickness greater than the maximum cross-sectional cell dimension can be used. For example, a slice thickness of 5-50 μm (e.g., 5 μm, 10 μm, 20 μm, 40 μm, or 50 μm) can be used, with a slice area of (2-4) × (2-4) cm, adhered to the fixed area of the microsphere chip. More preferably, the slice thickness is 8-12 μm (e.g., 8 μm, 9 μm, 10 μm, 11 μm, or 12 μm), with a slice area of (0.6-1) × (0.6-1) cm, adhered to the fixed area of the microsphere chip; multiple slices can also be obtained from a single biological sample. For example, multiple tissue slices can be obtained from a surgical biopsy sample by using a microtome blade to perform serial sectioning of the biopsy sample. In this way, spatial information between serial sections can be preserved, and the sections can be continuously analyzed to obtain three-dimensional information about the biological sample.

[0026] The fixative used in the present invention includes aldehydes such as formaldehyde, glutaraldehyde and paraformaldehyde, alcohols such as methanol, ethanol, and acetone, and the like, and the fixative can be used at any suitable level or concentration.

[0027] In some embodiments, the fixation may be terminated after fixation, and the fixation may be terminated using any suitable level or concentration of glycine and bovine serum albumin or other reagents or methods.

[0028] In some embodiments, the subsequent reaction can be performed directly without terminating the immobilization.

[0029] In some embodiments, the permeabilizing comprises permeabilizing the biological sample under chemical permeabilization conditions, enzymatic permeabilization conditions, or both.

[0030] In some embodiments, the chemical permeabilization conditions include contacting the biological sample with an alkaline solution, and the chemical permeabilization reagent includes one or more of Triton X-100 or Tween-20, Digitonin, NP40 or CA630; and also includes one or more of some organic solvents such as acetone, ethanol and methanol.

[0031] In some embodiments, the enzymatic permeabilization conditions comprise contacting the biological sample with an acidic solution comprising a protease. The enzyme comprises an aspartyl protease, preferably pepsin, a pepsin-like enzyme, or a functional equivalent thereof.

[0032] In some embodiments, protein inhibitors are also added during permeabilization to protect histones, including protease inhibitor complex (PIC), BSA, or functional equivalents thereof.

[0033] Preferably, the staining in step (2) includes any one of HE staining, DAPI staining or ssDNA staining.

[0034] In the present invention, for the convenience of visualization, biological sample can be stained using a variety of staining agents and staining techniques. In some embodiments, for example, the staining agent of any number and dosage can be used to stain the sample, including but not limited to acridine orange, Bismarck brown, carmine, Coomassie blue, cresyl violet, DAPI, eosin, ethidium bromide, acid fuchsin, hematoxylin, Hodgkin's stain, iodine, methyl green, methylene blue, neutral red, Nile blue, Nile red, osmium tetroxide, propidium iodide, rhodamine or saffron. Sample can be stained using hematoxylin-eosin (H&E) staining technique, Papanicolaou staining technique, Masson's trichrome staining technique, silver staining technique, Sudan red staining technique and / or periodic acid Schiff (PAS) staining technique. PAS staining is usually carried out after formaldehyde or acetone fixation. In some embodiments, sample can use Romanovsky staining, including Wright staining, Jenner staining, Kangrunwald staining, Leishman staining and Giemsa staining dyeing.

[0035] Preferably, the transposase in step (3) is linked to a special customized sequence.

[0036] Preferably, the special customized sequence has modifications, and the modifications include phosphorylation modification and / or biotin modification.

[0037] Preferably, the transposase comprises any one of Tn5 transposase, Mu transposase, a functional derivative of Tn5 transposase or a functional derivative of Mu transposase.

[0038] Preferably, the transposase simultaneously ligates two specially customized sequences to the gene fragment while cleaving the genome; the two specially customized sequences are double-stranded sequences, and the dsDNA is contacted with the transposome to generate more than one dsDNA fragment, each dsDNA fragment comprising a first 5' overhang and a second 5' overhang, wherein each dsDNA fragment in the more than one dsDNA fragment comprises a first strand comprising the first 5' overhang and a second strand comprising the second 5' overhang, wherein the transposome comprises the transposase, a first adaptor having the first 5' overhang, and a second adaptor having the second 5' overhang, wherein the second 5' overhang comprises a partial library sequencing adapter 2 sequence.

[0039] Preferably, the transfer in step (4) includes: transferring the fragmented genomic DNA to a chip with spatial information, and the transfer method includes active transfer or passive transfer; the active transfer includes applying an external electric field, and the passive transfer includes diffusion.

[0040] Preferably, the transfer includes any one of the following:

[0041] (1) First, the fragmented genomic DNA after the transposition reaction is hybridized with the bridging primer, and then the fragmented genomic DNA is connected to the capture probe of the chip with spatial information;

[0042] (2) The bridging primer, the capture probe of the chip with spatial information, and the fragmented genomic DNA after the transposition reaction are hybridized simultaneously.

[0043] Preferably, the length of the bridging primer is 10-30 bp, such as 10 bp, 15 bp, 20 bp, 25 bp or 30 bp.

[0044] Preferably, the bridging primer comprises a complementary sequence to at least a portion of the first 5' overhang.

[0045] Preferably, the DNA hybridization system in step (4) comprises: a bridging primer, a serine protease and a buffer.

[0046] Preferably, the system for the ligation and end-repair reaction in step (5) comprises: DNA polymerase, DNA ligase, buffer, dNTP and ATP.

[0047] Preferably, the DNA polymerase comprises any one of T4 DNA polymerase, T7 DNA polymerase, Klenow large fragment, phi29 DNA polymerase or Bst DNA polymerase.

[0048] Preferably, the DNA ligase comprises T4 ligase and / or T7 ligase.

[0049] Preferably, the denaturation and melting in step (6) comprises: performing a denaturation reaction using an alkaline melting solution.

[0050] Preferably, the alkaline melting solution includes KOH solution and / or NaOH solution.

[0051] Preferably, the concentration of the alkaline melting solution is 0.01-1 mM, such as 0.01 mM, 0.02 mM, 0.05 mM, 0.5 mM or 1 mM.

[0052] Preferably, the denaturation reaction time is 5-30 min, such as 5 min, 6 min, 8 min, 10 min, 20 min or 30 min.

[0053] The schematic diagram of the capture probe of the present invention is as follows Figure 1 shown. Figure 1 In Chinese: Read 1 refers to the Trueseq library read 1 primer (sequencing sequence 1 primer). The barcode sequence is necessary for spatial location information. The barcode sequence on each microsphere on the same chip is identical, ensuring that all genomic sequences from a cell carry the same barcode sequence, confirming that these sequences originate from the same cell. The barcode sequences on different microspheres are distinct, allowing them to be matched to the chip position for spatial location. The partial bridging primer complementary sequence is used to complement the bridging primer to capture fragmented genomic fragments.

[0054] In a second aspect, the present invention provides the use of the method for detecting spatial chromatin accessibility described in the first aspect in constructing a chromatin accessibility sequencing library.

[0055] Compared with the prior art, the present invention has the following beneficial effects:

[0056] (1) The method for detecting spatial chromatin accessibility described in the present invention can simultaneously perform staining imaging and ATAC sequencing analysis on the same tissue section. By analyzing chromatin accessibility, tissue imaging, and sequencing on the same section, the transcriptional activity state of chromatin can be reflected, which is an important direction for studying gene expression regulation.

[0057] (2) The spatial analysis of genomic DNA in biological samples using the method for detecting spatial chromatin accessibility described in the present invention can achieve simultaneous acquisition of information such as fluorescence staining and chromatin accessibility on the same tissue section. The experimental operation is simple, the repeatability is high, and the analysis results are more significant.

[0058] (3) The library synthesized using the method for detecting spatial chromatin accessibility described in the present invention is a Trueseq library. Only simple Trueseq sequencing is required to obtain the required barcode information, without the need for additional customized sequencing services. This method is low-cost and does not require complex microfluidic equipment. It is simple to operate and highly reproducible. It can be applied to the embryonic brain of young mice to map epigenetic mechanisms, which is of great significance in life science and biomedical research.

[0059] (4) The chip used in the method for detecting spatial chromatin accessibility of the present invention has long-sequence encoded silica microspheres. The microspheres are rich in variety, and primers with the same capture sequence are evenly distributed on the microspheres, which can capture chromatin accessible sequences, and can more comprehensively locate tissues, and then analyze the in situ information of gene expression of the epigenome on tissue sections. The present invention improves the chromatin accessible DNA hybridization, end repair and ligation reaction system, so that the chip and chromatin accessible DNA are fully in contact, which more effectively improves the capture efficiency of the chip and can obtain more information on chromatin open areas. BRIEF DESCRIPTION OF THE DRAWINGS

[0060] Figure 1 Schematic diagram of capture probe;

[0061] Figure 2 This is a graph analyzing the number of specific molecular tags in Example 1;

[0062] Figure 3 This is the tissue clustering statistical diagram in Example 1. DETAILED DESCRIPTION

[0063] To further illustrate the technical means and effects of the present invention, the present invention is further described below with reference to the embodiments and drawings. It should be understood that the specific embodiments described herein are only used to explain the present invention, rather than to limit the present invention.

[0064] If no specific techniques or conditions are specified in the examples, the experiments were carried out according to the techniques or conditions described in the literature in the field or according to the product instructions. If no manufacturer is specified for the reagents or instruments used, they are all conventional products that can be purchased through regular channels.

[0065] Example 1

[0066] This embodiment provides a kit for constructing a spatial single-cell ATAC genomics sequencing library, which can be used to construct a spatially organized in situ single-cell chromatin accessibility genomics sequencing library, and then the library can be used for sequencing analysis.

[0067] Specifically, this embodiment provides a single-cell genomics sequencing kit based on a spatial barcode chip and a transposase with specific double adapter processing, comprising:

[0068] (a) ATAC-omics chip with spatial barcode sequence information.

[0069] The synthesis method thereof refers to the preparation method of long coding sequence silica microspheres (CN216712064U; CN218710327U; CN117089599A; CN 115786457 A).

[0070] The ATAC omics chip includes a substrate and an anti-overflow protective film, wherein the substrate includes a plurality of microspheres, and each of the microspheres includes a probe;

[0071] The probe includes a sequencing primer fragment, a barcode fragment, a specific molecular index (UMI), and a capture structure for capturing fragmented genomic DNA, which are sequentially connected from the substrate upward; the capture structure is a partial bridging primer complementary sequence structure.

[0072] The sequences of the barcode fragment and the specific molecular tag include 40 randomly synthesized bases.

[0073] The anti-overflow protective film is a hydrophobic material film made of agarose condensed material and has a thickness of 40 μm.

[0074] (b) Transposase with a custom sequence attached.

[0075] The transposase is used to simultaneously ligate two special custom sequences onto gene fragments while cutting the genome, wherein the two special custom sequences are double-stranded sequences, and the double-stranded deoxyribonucleic acid (dsDNA) is contacted with the transposome to generate more than one dsDNA fragment, each dsDNA fragment comprising a first 5' overhang and a second 5' overhang, wherein each dsDNA fragment in the more than one dsDNA fragment comprises a first strand comprising the first 5' overhang and a second strand comprising the second 5' overhang, wherein the transposome comprises the transposase, a first adaptor having the first 5' overhang, and a second adaptor having the second 5' overhang, wherein the second 5' overhang comprises a portion of the Trueseq library sequencing adapter 2 sequence.

[0076] The transposase is Tn5 transposase, and the special customized sequence is modified with biotin.

[0077] (c) a bridging primer, comprising in sequence at least a portion of the complementary sequence of the first 5' overhang, for connecting the spatial chip and the fragment transposed by the transposase (such as Tn5), wherein the bridging primer sequence is 25 bp in length.

[0078] The preparation process of the transposase connected with a special customized sequence is as follows:

[0079] A first-strand primer of the first chain including the first 5' overhang and a second-strand primer of the second chain including the second 5' overhang were obtained, and the mixtures were respectively mixed with an ME sequence (specific site sequence for spatial chromatin accessibility analysis) in equal proportions, and then the temperature was lowered from 85°C to 20°C to obtain a transposase primer working solution; the transposase and the transposase primer working solution were incubated at 30°C for 60 minutes in the presence of an assembly buffer.

[0080] The kit further comprises a PCR amplification primer pair, wherein one of the upstream and downstream primers comprises a complementary sequence to the Trueseq library sequencing adapter 1 sequence, and the other comprises a complementary sequence to the Trueseq library sequencing adapter 2 sequence.

[0081] (d) Ligation and end-repair enzymes.

[0082] The kit provided in this embodiment also includes T4 DNA polymerase and T4 DNA ligase.

[0083] The kit also includes reagents for reverse transcription reaction and / or amplification reaction, tissue fixative (liquid), tissue permeabilizing agent (liquid), staining agent, enzyme digestion reaction solution, hybridization buffer, PCR buffer, nucleic acid purification reagent and PCR enzyme.

[0084] (e) The kit provided in this embodiment includes: an adhesive slide for transfer and a special clip for transfer.

[0085] Example 2

[0086] This example uses the kit for constructing the spatial single-cell ATAC genomics sequencing library described in Example 1.

[0087] 1. Tissue Fixation

[0088] (1) Remove the chip with human liver cancer tissue attached (shipped on dry ice) from -80℃ and place it on a 37℃ adapter with the tissue facing up and incubate for 1 minute (do not cover with a hot cover); if the tissue has just been attached in a freezing microtome, perform the same operation.

[0089] (2) Install the chip into a BMKMANU adapter (or without an adapter); add 100 μL of freshly prepared 0.2% formaldehyde to the tissue well to completely cover the tissue area and fix at room temperature for 10 minutes. After fixation, wash three times with 0.5 M Tris-HCl and 1× DPBS.

[0090] 2. Tissue Staining

[0091] Tissue fluorescence staining

[0092] Use the cell segmentation kit (BMKMANU) from Baichuang Zhizao and prepare the fluorescent reagent according to Table 1 below (vortex to mix, centrifuge briefly, and store in a dark place).

[0093] Table 1

[0094] Components Volume (μL) Enzyme-free water 82.5 Cell splitting buffer (vortex mixing, brief centrifugation) 27.5 Cell segmentation fluorescent reagent (vortex mixing, instant centrifugation) 0.55 Recombinant nuclease inhibitors 5.5 total 116.05

[0095] Place the chip on a horizontal surface, add 100 μL of cell segmentation fluorescent reagent to evenly cover the tissue to avoid bubbles, and incubate at room temperature in the dark for 5 minutes.

[0096] After the fluorescent reagent incubation is complete, discard any excess reagent and gently rinse the tissue five times in 0.1× SSC. Note that the OCT surrounding the tissue should have been rinsed clean by this point. If any residual OCT remains, increase the number of rinses as needed.

[0097] Use dust-free paper to wipe off excess liquid except for the tissue area, place it horizontally on a 37°C metal bath, and bake it away from light for 2 minutes to dry excess liquid on the tissue.

[0098] Tissue imaging

[0099] (1) Switch to dark field mode, place the chip on the carrier and load it into the instrument, and use a 40× objective lens to capture images.

[0100] (2) Select SpGreen and transflective channels, turn off automatic exposure, and select the SpGreen channel for both F and S.

[0101] (3) Select a 20× objective lens, use autofocus, and set the focusing frequency to 20.

[0102] (4) Flat Field Correction Selection: Strongest Statistics. Note: If the scan result shows uneven exposure, try Use Stored Comp Image.

[0103] (5) Select seamless mode.

[0104] (6) First, use the semi-transparent and semi-reflective channel to focus on the chip base plate, manually adjust the focus value and exposure time (you can try to set it to 5ms at the beginning), select the clearest value, the range is ±1, and the requirement is that the image can clearly see the dividing line of the chip base plate in both the organized and unorganized areas.

[0105] (7) Switch to the SpGreen channel and manually adjust the focus value and exposure time (try setting it to 25ms initially). During the left and right adjustment process, you can see the image going from blurry to clear and blurry. Select the clearest value within the range of ±1. The requirements are: the brightest part of the tissue should not be overexposed (should not appear bright white), and the darkest part should not be too dark (cell nuclei cannot be seen clearly). They need to be clearly distinguishable by the naked eye, as shown below. Note: During the focusing process, always be careful not to repeatedly adjust the same place to avoid quenching.

[0106] (8) Be sure to record the chip handle number corresponding to each sample, select the matrix where the tissue is located, select the entire matrix area, and start scanning.

[0107] 2.3. After staining, the cell nucleus is located and its boundaries are depicted based on fluorescence. This boundary information is mapped onto the chip, and sequencing data at the corresponding position is extracted to achieve accurate single-cell segmentation.

[0108] After the reaction, wash with 1× DPBS and remove any residual reagents.

[0109] 3. Cell Permeabilization

[0110] Add 100 μL of freshly prepared permeabilization reaction system (Table 2) to the tissue wells and incubate at room temperature for 10 min.

[0111] Table 2

[0112]

[0113]

[0114] After the reaction is complete, remove all remaining reagents by aspirating, wash with wash buffer for 10 minutes, and then remove all remaining reagents by aspirating.

[0115] The cleaning reaction system is shown in Table 3.

[0116] Table 3

[0117] Reagents volume 1MTris-HCl (PH7.4) 10 μL 5M NaCl 2μL <![CDATA[1MMgCl2]]> 3μL 5% BSA 200 μL Enzyme-free water 785μL total 1mL

[0118] 4. Cell Transposition

[0119] 4.1. Adapter preparation

[0120] The Tn5 naked enzyme used in this example was purchased from Vazyme.

[0121] (1) Primer name and sequence

[0122] Adaptor 1 (SEQ ID NO.01):

[0123] 5'phos-AGGCCAGAGCATTCGGTGTATAAGAGACAG, where phos represents phosphorylation.

[0124] Adaptor 2 (SEQ ID NO.02):

[0125] CAGACGTGTGCTCTTCCGATCTGTGTATAAGAGACAG.

[0126] ME primer (SEQ ID NO.03):

[0127] CTGTCTCTTATACAC.

[0128] Bridge oligo (SEQ ID NO.04):

[0129] 5'phos-CGAATGCTCTGGCCTCTCAAGCACGTGGAT, where phos stands for phosphorylation.

[0130] (2) Adaptor 1, Adaptor 2, and ME primer powders were dissolved in Annealing Buffer to 100 μM. The primer sequences were synthesized by Shanghai Bioengineering.

[0131] (3) Prepare the reaction systems shown in Table 4 respectively:

[0132] Table 4

[0133]

[0134] The reaction system can be scaled up proportionally according to actual needs.

[0135] (4) Vortex and vortex Reaction 1 and Reaction 2 to mix thoroughly, and briefly centrifuge to collect the solution at the bottom of the tube. Place in a PCR instrument and perform the following reaction program: After reacting at 85°C for 3 minutes, cool the temperature to 20°C at 1°C per minute.

[0136] (5) After the reaction is complete, mix equal volumes of Reaction 1 and Reaction 2. Add an equal volume of 100% glycerol and mix thoroughly. Name the mixture AdapterMix and store at -15°C.

[0137] 4.2. TTE Mix Assembly

[0138] (1) Add each reaction component to a sterilized PCR tube in sequence. The assembled reaction system is shown in Table 5:

[0139] Table 5

[0140]

[0141] (2) Use a pipette to gently pipette 20 times to mix thoroughly.

[0142] (3) Incubate at 30°C for 1 h. The reaction product is named TTE Mix and stored at -15°C.

[0143] The assembly buffer in the Tn5 transposon assembly system shown in Table 4 above was the assembly buffer provided by the supplier, and the assembly reaction was performed according to the standard operating procedure provided by the supplier. The transposition primer dimer is a double-stranded DNA structure.

[0144] DNA fragmentation

[0145] Add 100 μL of transposase mix to the tissue well and react at 37°C for 0.5 h.

[0146] The formula of 2×TD buffer is shown in Table 6.

[0147] Table 6

[0148] Reagents volume Dimethylformamide 20 μL 1M Tris-HCl pH 7.4 2μL <![CDATA[1MMgCl2]]> 1 μL 1× DPBS 77μL total 100 μL

[0149] The transposition reaction system is shown in Table 7.

[0150] Table 7

[0151] Reagents volume 2×TDbuffer 50μL 10% Tween-20 1 μL 0.5% Digitonin 2μL TTEMix 47μL total 100 μL

[0152] After the reaction, 50 mM EDTA was added to terminate the reaction at room temperature for 5 min, and then the plate was washed with 4×SSC and the residual reagent was removed.

[0153] 5. Transfer Hybridization

[0154] In the transfer cartridge, first add 10 μL of hybridization mix to the chip well with a specific chip slot, then stick the tissue well directly above the chip well so that the tissue well and the microsphere well are completely fitted together, and react in a 30°C wet box for 8 hours.

[0155] The hybridization reaction system is shown in Table 8.

[0156] Table 8

[0157] Reagents volume Bridging primer (100 μM) 3μL 1% Triton-X100 0.5μL 20 μg / μL Proteinase K 0.4μL 4×SSC buffer 6.1μL total 10 μL

[0158] After the reaction, wash with 1× DPBS and remove any residual reagents.

[0159] 6. End repair and connection

[0160] Add 100 μL of end repair reaction system and react at 18°C for 4 h.

[0161] The end repair reaction system is shown in Table 9.

[0162] Table 9

[0163] Reagents volume 10×buffer 10 μL Enzyme-free water 79μL 10mM dNTP Mixture 3μL T4 DNA polymerase 2μL T4 DNA ligase 5μL ATP 1 μL total 100 μL

[0164] After the reaction is completed, wash with Elution buffer for 2 minutes and remove the remaining reagents.

[0165] 7. Unchain

[0166] 45 μL KOH (0.08 M) was added to the tissue wells and melted at room temperature for 10 min; the supernatant was aspirated into a new PCR tube and neutralized by adding 6 μL Tris pH 7 (1 M).

[0167] 8. ATAC Library Preparation

[0168] The supernatant in the PCR tube in step 7 was added to the ATAC library reaction system shown in Table 10 for PCR amplification. The reaction program was as follows: 95°C for 30 s; 95°C for 15 s, 62°C for 20 s, 72°C for 1 min, 15 cycles; 72°C for 5 min, and stored at 4°C.

[0169] Table 10

[0170] Reagents volume 5×KAPAHiFibuffer 20 μL Each dNTP (10 μM) 3μL PrimerN5×× 4μL PrimerN7×× 4 μL KAPAHiFienzyme 2μL Enzyme-free water 16μL total 54μL

[0171] In this example, the Trueseq library Index primers were purchased from Vazyme.

[0172] The PCR products were purified using 0.8× magnetic beads, and the purified PCR products were directly sequenced using the Trueseq library.

[0173] 9. Data Analysis

[0174] The ATAC genomics data analysis process of the Baichuang Intelligent Manufacturing Space was used for analysis. Spatial barcoding technology was used to further perform spatial resolution sequencing of chromatin accessibility across the entire genome using technologies such as Tn5 translocation, and a spatial distribution map of chromatin open region information was obtained ( Figure 2 ) and cluster analysis diagram ( Figure 3 ), combines the ability to analyze chromatin accessibility with the spatial location of cells. By mapping the location of cell types to their native tissue context, we can directly visualize cell types in tissues based on their overall epigenetic state. Figure 2 and Figure 3 It can be seen that the spatial distribution of the number of specific molecular labels and the number of genes of tumor tissue is consistent with its tissue morphology. Unsupervised clustering identified 12 major spatial clusters from 0 to 11, revealing different spatial types within tumor tissue, which is consistent with the histological structure and reflects the complex regionalization of this tissue.

[0175] In summary, the method of the present invention can simultaneously perform staining imaging and chromatin accessibility sequencing analysis on the same tissue section. Chromatin accessibility sequencing analysis can reflect the transcriptional activity state of chromatin, which is an important direction for studying gene expression regulation. It plays an important role in epigenetic mapping, cell differentiation and development, and the occurrence and development of various diseases. Without the need for complex microfluidic equipment, staining imaging and chromatin accessibility information can be simultaneously obtained on the same tissue section. The experimental operation is simple, the repeatability is high, and the analysis results are more significant.

[0176] The applicant states that the present invention is intended to illustrate the detailed methods of the present invention through the above-described embodiments, but the present invention is not limited to the above-described detailed methods, that is, it does not mean that the present invention must rely on the above-described detailed methods in order to be implemented. Those skilled in the art should understand that any improvements to the present invention, equivalent substitutions for various raw materials in the products of the present invention, addition of auxiliary ingredients, and selection of specific methods, etc., are all within the scope of protection and disclosure of the present invention.

Claims

1. A method for detecting spatial chromatin accessibility, characterized in that The method comprises the following steps: (1) Slice the tissue sample to be tested on a chip with spatial information; (2) Fixing, permeabilizing, and staining the tissue samples to be tested; (3) mixing the permeabilized and stained tissue sample with the transposon sequence inserted and the transposase to obtain fragmented genomic DNA; (4) DNA hybridization based on transfer mode to capture fragmented genomic DNA; (5) using DNA polymerase and DNA ligase to perform ligation and end-repair reactions on the fragmented genomic DNA; (6) denaturation and melting to obtain fragmented genomic DNA with spatial information; (7) Sequencing analysis.

2. The method for detecting spatial chromatin accessibility according to claim 1, characterized in that The chip with spatial information in step (1) comprises a substrate, the substrate carries microspheres, and each microsphere carries a capture probe; the capture probe comprises a sequencing primer fragment, a barcode fragment, a specific molecular tag, and a capture structure for capturing fragmented genomic DNA, which are sequentially connected from the substrate upward; Preferably, the chip with spatial information contains an anti-overflow protective film; Preferably, the anti-overflow protective film has a thickness of 10 μm-100 μm; Preferably, the capture probes on the same microsphere in the chip with spatial information contain the same spatial barcode; Preferably, the capture probe is modified; Preferably, the modification includes any one of phosphorylation modification and biotin modification.

3. The method for detecting spatial chromatin accessibility according to claim 1 or 2, characterized in that The staining in step (2) includes any one of HE staining, DAPI staining or ssDNA staining.

4. The method for detecting spatial chromatin accessibility according to any one of claims 1 to 3, characterized in that The transposase in step (3) is connected to a special customized sequence; Preferably, the special customized sequence has modifications, and the modifications include phosphorylation modification and / or biotin modification; Preferably, the transposase comprises any one of Tn5 transposase, Mu transposase, a functional derivative of Tn5 transposase or a functional derivative of Mu transposase; Preferably, the transposase simultaneously ligates two specially customized sequences to the gene fragment while cutting the genome; the two specially customized sequences are double-stranded sequences, and the dsDNA is contacted with the transposome to generate more than one dsDNA fragment, each dsDNA fragment comprising a first 5' overhang and a second 5' overhang, wherein each dsDNA fragment in the more than one dsDNA fragment comprises a first strand comprising the first 5' overhang and a second strand comprising the second 5' overhang, wherein the transposome comprises the transposase, a first adaptor comprising the first 5' overhang, and a second adaptor comprising the second 5' overhang, wherein the second 5' overhang comprises a partial library sequencing adapter 2 sequence.

5. The method for detecting spatial chromatin accessibility according to any one of claims 1 to 4, characterized in that The transfer in step (4) includes: transferring the fragmented genomic DNA to a chip with spatial information, and the transfer method includes active transfer or passive transfer; the active transfer includes applying an external electric field, and the passive transfer includes diffusion; Preferably, the transfer includes any one of the following: (1) First, the fragmented genomic DNA after the transposition reaction is hybridized with the bridging primer, and then the fragmented genomic DNA is connected to the capture probe of the chip with spatial information; (2) The bridging primer, the capture probe of the chip with spatial information, and the fragmented genomic DNA after the transposition reaction are hybridized simultaneously.

6. The method for detecting spatial chromatin accessibility according to claim 5, characterized in that The length of the bridging primer is 10-30 bp; Preferably, the bridging primer comprises a complementary sequence to at least a portion of the first 5' overhang.

7. The method for detecting spatial chromatin accessibility according to any one of claims 1 to 6, characterized in that The DNA hybridization system in step (4) includes: a bridging primer, a serine protease and a buffer.

8. The method for detecting spatial chromatin accessibility according to any one of claims 1 to 7, characterized in that The system for the ligation and end repair reaction in step (5) includes: DNA polymerase, DNA ligase, buffer, dNTP and ATP; Preferably, the DNA polymerase comprises any one of T4 DNA polymerase, T7 DNA polymerase, Klenow large fragment, phi29 DNA polymerase or Bst DNA polymerase; Preferably, the DNA ligase comprises T4 ligase and / or T7 ligase.

9. The method for detecting spatial chromatin accessibility according to any one of claims 1 to 8, characterized in that The denaturation and melting in step (6) includes: using an alkaline melting solution to perform a denaturation reaction; Preferably, the alkaline melting solution comprises KOH solution and / or NaOH solution; Preferably, the concentration of the alkaline melting solution is 0.01-1 mM; Preferably, the denaturation reaction time is 5-30 minutes.

10. Use of the method for detecting spatial chromatin accessibility according to any one of claims 1 to 9 in constructing a chromatin accessibility sequencing library.

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