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WO2024012418A1 - Procédé de capture de conformation tridimensionnelle de chromatine et son utilisation - Google Patents

Procédé de capture de conformation tridimensionnelle de chromatine et son utilisation Download PDF

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WO2024012418A1
WO2024012418A1 PCT/CN2023/106632 CN2023106632W WO2024012418A1 WO 2024012418 A1 WO2024012418 A1 WO 2024012418A1 CN 2023106632 W CN2023106632 W CN 2023106632W WO 2024012418 A1 WO2024012418 A1 WO 2024012418A1
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chromatin
spatial
sequence
barcode
ligation
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陈雪鹏
陈振平
喻昊
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Guangzhou National Laboratory
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Guangzhou National Laboratory
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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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6869—Methods for sequencing
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B20/00—ICT specially adapted for functional genomics or proteomics, e.g. genotype-phenotype associations
    • G16B20/20—Allele or variant detection, e.g. single nucleotide polymorphism [SNP] detection
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B30/00—ICT specially adapted for sequence analysis involving nucleotides or amino acids
    • G16B30/20—Sequence assembly
    • G—PHYSICS
    • G16—INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR SPECIFIC APPLICATION FIELDS
    • G16B—BIOINFORMATICS, i.e. INFORMATION AND COMMUNICATION TECHNOLOGY [ICT] SPECIALLY ADAPTED FOR GENETIC OR PROTEIN-RELATED DATA PROCESSING IN COMPUTATIONAL MOLECULAR BIOLOGY
    • G16B50/00—ICT programming tools or database systems specially adapted for bioinformatics

Definitions

  • the invention belongs to the field of genomics, and specifically relates to chromatin three-dimensional conformation capture technology and its application.
  • the 2-meter-long chromatin composed of DNA and proteins is highly compressed to fit into a limited space that only varies between 5-10 ⁇ m in diameter.
  • Hi-C technology research based on "proximity joining" has revealed that the genome is a highly hierarchical organization in the nucleus of eukaryotes, called the three-dimensional conformation of chromatin. The importance of the three-dimensional conformation of chromatin in the study of genome function has received widespread attention.
  • Dynamic changes in the three-dimensional conformation of chromatin play an important role in mouse limb development, muscle progenitor cell specialization and myogenic differentiation, cerebral cortex development, and the development and differentiation of dendritic cells, and are also responsible for various organ developmental malformations and Cancer has been reported to be associated with abnormalities in the three-dimensional conformation of chromatin.
  • the high-throughput capture method of chromatin three-dimensional conformation at the spatial omics level is still blank.
  • the reason is due to various technical limitations: (1) The existing Hi-C technology at the cell population level (in situ Hi-C , tag Hi-C, etc.) requires the operation of extracting cell nuclei first, which cannot be achieved on tissue sections; (2) Since the chromatin connection products produced during the Hi-C experiment are complexes containing proteins, Hi at the cell population level When -C (in situ Hi-C, tag Hi-C, etc.) is used to interrupt chromatin, the cell nucleus needs to be decross-linked first to expose the DNA so that subsequent disruption can achieve full genome coverage; while in tissue On slices, direct decross-linking reaction without spatial positioning labeling will destroy the cell structure, release DNA into the solution, and lose the position information of cells in the tissue; (3) In situ genome sequencing technology (IGS) ) Using imaging to infer the structure of chromatin not only requires the support of high-resolution imaging microscopes, but also currently can only achieve DNA
  • the present invention performs a chromatin proximity ligation reaction in situ, and labels the proximity ligation products with Barcode combinations specific to spatial position information, thereby achieving the capture of the three-dimensional conformation of chromatin under the condition of retaining spatial resolution.
  • the present invention provides a spatial chromatin analysis method, a spatial chromatin capture method or a spatial chromatin high-throughput sequencing library construction method.
  • the method includes the following steps:
  • step (3) Use the Tn5 transposase complex to digest and fragment the circular chromatin proximity ligation product obtained in step (2) to obtain chromatin proximity ligation product fragments, and at the same time, add Adapter to obtain chromatin adjacent ligation product fragments with adapters;
  • step (2) the location where the chromatin proximity ligation reaction occurs is labeled with a label.
  • the label is biotin.
  • restriction enzymes are used to fragment chromatin within the cell.
  • restriction enzymes are used to fragment intracellular chromatin to obtain sticky ends.
  • restriction endonuclease can be any restriction endonuclease, including but not limited to MboI, and can also be DNase enzyme, MNase enzyme, Tn5 transposase and other restriction endonucleases.
  • the intracellular chromatin is fragmented to obtain sticky ends in step (1)
  • the sticky ends of the chromatin are blunted, and biotin-labeled bases are introduced to obtain biotin-labeled bases. Blunt end fragments.
  • the cells in step (1) and the nuclei within the cells are permeabilized. reason.
  • the present invention uses a different cell permeabilization solution from the traditional Hi-C experiment to achieve permeabilization of cells and nuclei in the tissue slices in situ, so that macromolecular reagents, such as enzymes, in subsequent experimental reactions can enter In situ reactions are achieved in the cell nucleus. Therefore, reagents that can permeabilize cell nuclei can be selected as needed.
  • the cells in step (1) are cells in a tissue section.
  • the spatial chromatin analysis method can be performed in situ on tissue sections.
  • step (4) through a ligation reaction, the chromatin with the adapter obtained in step (3) is adjacent to the 5' or 3' end of the ligation product fragment, and the spatial position-specific lateral markers and longitudinal markers are connected. .
  • step (4) through a ligation reaction, the spatial position-specific lateral markers Barcode H and Vertical marking Barcode V.
  • step (4) through a ligation reaction, the Barcode H ligation complex and Barcode V are added to the 5' end or 3' end of the ligated chromatin adjacent to the ligation product fragment obtained in step (3). Junction complex.
  • the Barcode H linker complex contains Linker H and Barcode H domains.
  • the Linker H has the following structure:
  • linker1-R contains part or all of the complementary sequence of linker1;
  • the Barocde H domain has the following structure:
  • linker2-R contains part or all of the complementary sequence of Linker2,
  • the Barcode Hi is a DNA sequence with a length of 3-50 bp, which is used to locate the position of the lateral channel where the Reads are located.
  • i represents the number of channels, which can be 4-40,000.
  • the Barcode V linker complex contains Linker V and Barcode V domains.
  • the Linker V has the following structure:
  • linker3-R contains part or all of the complementary sequence of linker3;
  • the Barcode V domain has the following structure:
  • the linker4-R contains part or all of the complementary sequence of linker4, and the Barcode Vj is a DNA sequence with a length of 3-50 bp, which is used to locate the position of the longitudinal channel where the Reads are located.
  • i represents the number of channels, which can be 4 -40000 items,
  • the Primer can be a sequencing primer, used for on-machine sequencing of the library, or a Linker5 sequence, used for the next round of ligation reaction.
  • step (3) adding an adapter to the chromatin proximity ligation product fragment can be compatible with the subsequent required spatial position tag sequence Barcode H and Barcode V ligation.
  • tagHi-C method also uses Tn5 transposase for fragmentation to avoid ultrasonic interruption of DNA
  • tagHi-C directly uses Tn5 transposase to connect DNA sequencing library adapters and cannot be connected with subsequent The required spatial position tag sequences Barcode H and Barcode V are connected and compatible, so tagHi-C cannot capture the three-dimensional conformation of chromatin while retaining spatial resolution.
  • Barcode H domain has the following sequence:
  • Barcode V domain has the following sequence:
  • the Linker H has the following sequence:
  • the Linker V has the following sequence:
  • the method further includes the step of using magnetic beads to enrich labeled fragments after step (4), and the label is the position where the chromatin proximity ligation reaction occurs with the marker in step (2). Marking made.
  • the method further includes the step of enriching the biotin-labeled fragments using magnetic beads.
  • the method further includes the step of treating the chromatin with HCl after the chromatin proximity ligation reaction in step (2) to remove histones from the chromatin.
  • microfluidic chip labeling technology is used to connect the spatial location information in step (4).
  • Information-specific Barcode sequence markers are used to connect the spatial location information in step (4).
  • the width of the microfluidic channel in the microfluidic chip is 5-500 ⁇ m, such as 10 ⁇ m, 20 ⁇ m, 25 ⁇ m, 30 ⁇ m, 40 ⁇ m or 50 ⁇ m.
  • the number of microfluidic channels in the microfluidic chip is 4-40,000 channels, for example, it can be 12 channels, 24 channels, 36 channels, 48 channels, 60 channels, 72 channels, 84 channels or 96 channels.
  • the number of microfluidic channels can be selected as needed to achieve coverage of tissue sections of different area sizes.
  • the Tn5 transposase complex includes a Tn5 transposase and a linker sequence that enables subsequent efficient ligation of spatial position information-specific Barcode sequence tags at the 5' or 3' end through a ligation reaction.
  • the adapter sequence includes Adaptor1 and Adaptor2, which can realize efficient connection of spatial position-specific lateral markers and longitudinal markers at the 5' or 3' end through subsequent ligation reactions.
  • the horizontal mark is Barcode H.
  • the vertical mark is Barcode V.
  • the Tn5 transposase complex also contains a ME sequence.
  • the Tn5 transposase complex includes a Tn transposase and a linker sequence
  • the linker sequence includes a sequence matching the Barcode sequence connected in step (4).
  • the adapter sequences include Adaptor1 and Adaptor2.
  • the Tn5 transposase complex further comprises a ME sequence.
  • the Adaptor1 has the following structure:
  • the ME-R is complementary to the ME sequence and forms a complementary structure when annealed to the ME sequence, and linker1 is used to connect with the Barcode H connection complex.
  • the Adapter2 has the following structure:
  • the ME-R is complementary to the ME sequence and forms a complementary structure when annealed to the ME sequence.
  • the Adaptor1 domain has the following sequence:
  • the Adapter2 domain has the following sequence:
  • the ME sequence has the following sequence:
  • spatial position-specific horizontal markers and vertical markers are efficiently connected only at the 5' or 3' end, which can avoid the tandem structure generated during subsequent PCR reactions and achieve efficient amplification of the library.
  • the method includes the steps of:
  • step (f) Use the product obtained in step (e) with HCl to remove histones from the chromatin, and then use the Tn5 transposition complex to cut off the chromatin DNA fragments in the nuclei of the sliced cells, and add different adapters to both ends (one end It is the sequencing adapter Adapter2, and one end is the adapter Adapter1 for connecting Barcode);
  • step (g) Overlay the tissue slices treated with Tn5 transposase in step (f) with the lateral microfluidic chip to form a lateral channel on the surface where the tissue slices and the microfluidic chip are combined, and add Mix the ligation mixture with different types of Barcode H to perform the ligation reaction;
  • step (h) Overlay the tissue slices processed in step (g) with the longitudinal microfluidic chip, form a longitudinal channel on the surface where the tissue slices and the microfluidic chip are combined, and add a mixture of different types of
  • the connection mixture of Barcode V is used for connection reaction.
  • the intersection point of the horizontal and vertical channels is the pixel, and the total number of pixels is the number of horizontal channels multiplied by the number of vertical channels;
  • step (i) using a decrosslinking buffer to perform a decrosslinking reaction on the tissue section processed in step (h);
  • the primer of the Barcode V domain is a linker5 sequence
  • the tissue sections are subjected to more rounds of ligation reactions, and the more rounds of ligation reactions are using Barcode S ligation complex. Label multiple slices of the same sample,
  • the Barcode S junction complex contains the following sequence:
  • the linker5R contains part or all of the complementary sequence of Linker5,
  • Barcode S domain [Sequencing Primer]-[Barcode Sk]-[Linker6R],
  • the sequencing primer is a library on-machine sequencing primer;
  • the Barcode Sk is a DNA sequence with a length of 3-50 bp, which is used to determine the number of the slice.
  • the present invention provides a spatial chromatin high-throughput sequencing method, which method includes the steps of the spatial chromatin high-throughput sequencing library construction method of the first aspect, and the step of sequencing.
  • the method further includes the step of HE staining adjacent sections to demonstrate morphological results.
  • the present invention provides a method for preparing a three-dimensional conformation map of spatial chromatin in a multi-dimensional organization.
  • the method includes the steps in the spatial chromatin capture method of the first aspect, and connecting the spatial position information in step (4).
  • multiple rounds of ligation reactions are performed on each slice to mark different slices of the same sample, different samples, etc.
  • the Primer sequence of Barcode V is a Linker5 sequence, and tissue slices can be subjected to more rounds of ligation reactions.
  • the ligation reaction such as adding an additional round of Barcode S to label multiple slices of the same sample, enables the capture of the three-dimensional conformation of chromatin in the three-dimensional space of the 3D tissue.
  • the method can realize the preparation of a three-dimensional spatial chromatin conformation map of a complete tissue at the three-dimensional level.
  • Subsequent library construction experiments can be carried out with multiple slices in the same tube, saving reagents and time costs in the experiment; the obtained sequencing results are based on the picture of each pixel position information and the order of each slice, using image stitching technology Merge and splice to obtain the spatial chromatin three-dimensional conformation at the three-dimensional 3D level of the tissue.
  • the present invention provides a method for preparing a spatial multi-omics map, which method includes the steps of the spatial chromatin capture method of the first aspect, and the step of using a poly T primer with a linker to perform reverse transcription, And after connecting the Barcode sequence markers with specific spatial position information in step (4), multiple rounds of ligation reactions are performed on each slice to achieve labeling of different slices, different samples, etc.
  • Subsequent library construction experiments can be carried out with multiple slices in the same tube, thereby saving reagents and time costs in the experiment; the obtained sequencing results are based on the picture of each pixel position information and the order of each slice, using image stitching technology Merge and splice to obtain the three-dimensional spatial chromatin conformation or spatial transcriptome map of the tissue at the three-dimensional 3D level, and realize the three-dimensional chromatin conformation and transcription of cells in different regions of the tissue. annotation of group information, thereby discovering more regulatory mechanisms at the chromatin level and their spatial location relationships during cell fate decisions and differentiation processes.
  • the present invention provides a method for analyzing a spatial chromatin high-throughput sequencing library, which includes the steps of the spatial chromatin high-throughput sequencing library construction method of the first aspect.
  • the method also includes the following steps:
  • the present invention provides the application of the methods of the first to fifth aspects in high-throughput sequencing of spatial chromatin.
  • the present invention realizes the capture of the three-dimensional conformation of chromatin under the condition of retaining spatial resolution and fills in the space group. There is a lack of technical gaps in capturing three-dimensional conformational information of chromatin at the scientific level.
  • the present invention uses microfluidic chip labeling technology to label the three-dimensional conformation of chromatin with position information. It is cheap, easy to operate, and has good scalability.
  • the width of the microfluidic channel can be adjusted to achieve different resolutions (5-500 ⁇ m, etc.) of spatial position information marking; in addition, the microfluidic channel can be flexibly customized. quantity to achieve coverage of tissue sections of different area sizes.
  • the number of Barcode H can be selected according to the number of microfluidic channels.
  • the present invention can further treat tissue slices with HCl to remove histones on the chromatin to expose the DNA, ensuring unbiased cutting of the Tn5 transposition complex, and achieving complete genome analysis on the tissue slices. Unbiased capture of chromatin three-dimensional conformational information.
  • the composition sequence of the Tn5 transposase complex is improved during the chromatin fragmentation process, and different linkers are added to both ends of the DNA fragment, so that the subsequent ligation reaction can efficiently connect spatial position information markers only at one end, avoiding subsequent
  • the tandem structure formed during the PCR amplification reaction ensures the correct library structure.
  • the present invention can also be combined with the spatial transcriptome to realize spatial multi-omics data capture.
  • the poly T primer containing part or all of the Adapter sequence is used to perform reverse transcription on the slice, so that the mRNA sequence is reverse transcribed into a barcode with subsequent connection.
  • Adapter sequence when performing the library construction step of spatial chromatin three-dimensional structure, you can add Barcode to cDNA and chromatin DNA at the same time.
  • Simultaneous implementation of spatial multi-omics can combine gene expression and regulatory mechanisms in the same cell for in-depth analysis, and more accurately study the regulatory mechanism of gene expression.
  • the present invention can capture different tissue substructures and even the three-dimensional chromatin structure of a single cell on a slice, and can be applied to the detection of chromatin conformational variation in clinical samples with very precious sample quantities.
  • Hi-C High-throughput chromosome conformation capture
  • the specific experimental steps are to crosslink the cells with formaldehyde, extract the nucleus and conduct nuclear membrane analysis. Permeabilize, use restriction endonuclease to digest chromatin DNA in the nucleus to generate sticky ends; sticky ends are labeled with biotin and proximity ligation reaction, and finally undergo cross-linking de-crosslinking, DNA fragmentation, and magnetic beads enrichment containing biotin The ligated fragments were finally subjected to library construction and paired-end sequencing.
  • Hi-C technology can achieve unbiased three-dimensional chromatin conformation capture at the whole genome level
  • traditional Hi-C technology is based on the cell population level and cannot achieve unbiased three-dimensional chromatin conformation with spatial resolution on tissue sections. Capture of conformational regulatory information.
  • the present invention achieves in-situ chromatin proximity ligation reaction on tissue sections, and on this basis, spatially position-specific Barcode H and Barcode V labeling of proximity ligation products, thereby enabling chromatin proximity ligation products to be labeled
  • the spatial position information in the tissue is traced back and analyzed to obtain the three-dimensional conformation information of chromatin under the condition of retaining spatial resolution.
  • the traditional Hi-C experiment is based on cell populations.
  • the cell nuclei need to be extracted.
  • the cells on the tissue sections are all adhered to the glass slides, making it impossible to extract the cell nuclei. Therefore, the present invention uses the same method as traditional Hi-C.
  • Experiment with different cell permeabilization solutions to achieve permeabilization of cells and nuclei in tissue sections in situ so that macromolecular reagents in subsequent experimental reactions, such as enzymes, can enter the cell nucleus to achieve in situ reactions;
  • tagHi-C Although the tagHi-C method also uses Tn5 transposase for fragmentation to avoid ultrasonic disruption of DNA, tagHi-C directly uses Tn5 transposase to connect DNA sequencing library adapters, resulting in two fragmented products. Both ends are connected to Illumina sequencing adapters, which are incompatible with the subsequent ligation reaction of the required spatial position tag sequences Barcode H and Barcode V. Therefore, tagHi-C cannot capture the three-dimensional conformation of chromatin while retaining spatial resolution;
  • the present invention first performs cell permeabilization and proximity ligation reactions on slices to fix and preserve the three-dimensional conformation state of chromatin in the cell nucleus. Then, after HCl treatment and Tn5 transposase digestion, the chromatin proximity ligation product DNA fragments are ionization, and one end of the DNA adjacent to the ligation product is equipped with an Adapter sequence that can be used for subsequent ligation reactions, and the other end is equipped with a sequencing adapter sequence, and then through the ligation reaction, spatial position specificity is achieved at the 5' or 3' end of the DNA adjacent to the ligation product.
  • the horizontal marker Barcode H and the vertical marker Barcode V are connected.
  • magnetic beads are used to enrich the biotin-labeled fragments, and PCR amplification is used to build the library.
  • connecting a spatial position label to only one end of the DNA can avoid the tandem structure produced during subsequent PCR reactions and achieve efficient amplification of the library;
  • the present invention can perform spatial chromatin three-dimensional conformation capture experiments on tissue sections. After adjusting the Barcode sequence markers with specific spatial position information, each slice is subjected to multiple rounds of ligation reactions to mark different slices of the same sample, different samples, etc.
  • the Primer sequence of Barcode V is a Linker5 sequence
  • tissue slices can also Perform more rounds of ligation reactions, such as adding an additional round of Barcode S to label multiple slices of the same sample to capture the three-dimensional conformation of chromatin in the three-dimensional space of the 3D tissue.
  • spatial Hi-C refers to high-throughput capture of spatial chromatin conformation, or high-throughput capture of three-dimensional chromatin conformation, or capture of three-dimensional spatial chromatin conformation.
  • the bacterial transposase Tn5 exploits the unique "tagging" function of dimeric Tn5, which cleaves double-stranded DNA (dsDNA) and ligates the resulting DNA ends to specific adapters.
  • dsDNA double-stranded DNA
  • Genetically engineered Tn5 can be widely used in sequencing library preparation due to its rapid synthesis capability and low sample input.
  • Tn5 reacts directly with dsDNA, breaking double-stranded DNA in one step and adding adapters to both ends of the DNA. This simple one-step labeling reaction greatly simplifies the experimental process, shortens the working time and reduces the cost, and is widely used in DNA sequencing library construction technology.
  • the entire Tn5 transposon sequence is not necessary for transposition. As long as the terminal core sequence of the transposon is needed, the transposase can insert this part of the sequence into the genome. Based on this principle, the sequencing adapter sequence is added to the terminal core sequence to transpose. The enzyme cleaves double-stranded DNA, and then connects the adapter sequences to both ends of the DNA fragment to complete library construction.
  • the traditional Illumina NEB library construction method requires DNA fragmentation, end repair, adapter ligation, library amplification, multiple purification and sorting steps, etc., which is time-consuming.
  • transposase construction of NGS libraries can fragment DNA and Multi-step reactions such as end repair and adapter ligation are simplified into one-step reactions, which greatly shortens the library construction time and improves work efficiency.
  • Single-cell sequencing can unbiasedly define cell types and states, but it cannot obtain information on the spatial distribution of biomolecules and cells in tissues.
  • the emergence of spatial transcriptomics makes up for this shortcoming of single-cell sequencing, allowing us to describe cellular functions and states in natural tissue environments.
  • Figure 1A, Figure 1B, Figure 1C and Figure 1D show the high-throughput capture experimental flow chart of spatial chromatin of the present invention.
  • Figure 2 shows a schematic diagram of library construction for spatial chromatin high-throughput sequencing of the present invention.
  • Figure 3 shows the library fragment distribution of mouse E13.5 day tissue sections obtained by Spatial Hi-C technology.
  • Figure 4A and Figure 4B show the different types of each pixel in mouse E13.5 day tissue sections in the embodiment of the present invention.
  • Reads distribution ( Figure 4A) Violin plot of Raw reads distribution ( Figure 4B) Violin plot of Cis_long range contact (>20kb) distribution.
  • Figure 5A, Figure 5B, Figure 5C and Figure 5D show the analysis results of the Spatial Hi-C library of mouse E13.5 day tissue sections in the embodiment of the present invention:
  • Figure 5A UMAP dimensionality reduction clustering results
  • Figure 5B Clustering Map of class analysis results
  • Figure 5C Map example 1 of the spA/B value of a single bin on the tissue section
  • Figure 5D Example 2 of the map of the spA/B value of a single bin on the tissue section.
  • Figure 6 shows the joint analysis of the A/B values of the pixels in the liver region in the mouse E13.5 day tissue sections and the single-cell transcriptome in the embodiment of the present invention.
  • Example 1 Utilizing the Spatial Hi-C method of the present invention to capture the spatial chromatin three-dimensional conformation of mouse E13.5 embryos, including the following steps:
  • mice on day E13.5 were sacrificed by cervical dislocation, the embryos were taken out, put into pre-cooled PBS buffer on ice, and the placenta and other tissues were removed under a stereomicroscope. After removing the mouse embryos, wash them once with PBS and put them into an OCT embedding box. After absorbing the residual liquid on the surface of the embryos, add pre-cooled OCT on ice to fill the embedding box and place it at -80°C overnight. Take out the embedded mouse embryos from -80°C, balance them in a freezing microtome for 30 minutes and then slice them. Place the tissue sections on adhesive glass slides and store them in a -80°C refrigerator.
  • tissue sections were first cross-linked (400 ⁇ L of 1% formaldehyde solution, cross-linked at room temperature for 10 min), and then 2.5 MGlycine (32.5 ⁇ L, final concentration of 0.125 M, treated at room temperature for 5 min) was added to terminate the cross-linking.
  • Preparation of the transposition complex (1) Assemble the adapter: Dissolve the ME sequence (5Phos/CTGTCTCTTATACACATCT/3ddC), Adapter 1 (5Phos/CGCGCTGCATACTTGAGATGTGTATAAGAGACAG) and Adapter2 (GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG) respectively in lysis buffer (40mM Tris-HCl (PH8.0) ), 50mM NaCl) at a concentration of 100 ⁇ M. Mix the ME sequence (10 ⁇ L) with equal volumes of adapter sequence 1 and adapter sequence 2 (10 ⁇ L) respectively, mix thoroughly and then anneal: 95°C.
  • Transposome assembly 5 ⁇ L novoNGS Tn5 transposase (10 pmol/ ⁇ L), 1 ⁇ L adapter mixture, mix thoroughly, and react at 23°C for 30 minutes.
  • Barcode connection complex by labeling the spatial position of chromatin DNA: Add 5 ⁇ L Barcode H (5Phos/CATCGGCGTACGACT[BarcodeHi]ATCCACGTGCTTGAG, BarcodeHi is an 8 bp Barcode sequence, composed of four deoxynucleotides of ATCG) (100 ⁇ M) in a PCR tube ), where the type of BarcodeHi depends on the number of microfluidic channels.
  • Barcode H 5 ⁇ L Linker1H (100 ⁇ M) (CAAGTATGCAGCGCGCTCAAGCACGTGGAT), 10 ⁇ L 2X annealing buffer are used. After mixing, denature at 95°C for 5 minutes.
  • Barcode Vj (100 ⁇ M) (TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG[BarcodeVj]GTGGCCGATGTTTCG BarcodeVj is an 8bp Barcode sequence, composed of four deoxynucleotides ATCG) (100 ⁇ M) in the PCR tube, where The type of BarcodeVj depends on the number of microfluidic channels. In this example, 50 types of Barcode H), 5 ⁇ L Linker V (100 ⁇ M) (AGTCGTACGCCGATGCGAAACATCGGCCAC), 10 ⁇ L 2 ⁇ annealing buffer are used. After mixing, denature at 95°C for 5 minutes. , -0.1°C/s cooling to 20°C. The ratio of the 2 ⁇ annealing buffer is shown in Table 7 below.
  • SPRI beads (Beckman) added to the 0.7-fold reaction system were used to recover DNA of more than 300 bp, and sequenced using the Illumina NovaSeq system.
  • the results of the cluster analysis are mapped on the tissue section pictures according to the pixel position, and further analyzed based on the morphological characteristics.
  • the present invention is applied to perform SpatialHi-C experiments on mouse E13.5 embryonic slices (the process is shown in Figure 1A, Figure 1B, Figure 1C and Figure 1D), and a library is constructed (the structure is shown in Figure 2).
  • the distribution of library fragments is shown in Figure As shown in 3, it is between 300-1500bp.
  • the illumina novoseq platform was used for sequencing, and a total of 485,962,701 pairs of raw reads were obtained.
  • the obtained sequencing data is analyzed using the analysis process of the present invention.
  • the Reads that correctly connected the two rounds of Barcode were 445, 539, 708 pairs, accounting for 91.68%; among them, the Reads that could be mapped to the mouse reference genome were 421, 562, 824 pairs, accounting for 94.61 %.
  • HiCExplorer analysis was used to effectively capture 143, 102, 598 pairs of reads containing chromatin three-dimensional conformation information (Valid reads), accounting for 29.44% of total reads; among them, long-distance interactions within chromosomes (Cis_long range>20kb contact) Accounting for 45.05% of the total valid reads.
  • the sequencing data was split into separate data for each pixel according to the Barcode combination, resulting in 2353 data sets.
  • the effective number of pixels accounts for 94.12% of the total number of pixels.
  • Higashi was used for cluster analysis to obtain 5 categories (Figure 5A). These 5 categories of pixels were displayed in situ on the slices ( Figure 5B). It can be seen that different categories have obvious position specificity and are related to the different tissue types of tissue sections.
  • SpatialHi-C can efficiently capture the three-dimensional image of chromatin with spatial resolution.
  • each pixel-specific A/B compartment is calculated to represent the open state of chromatin (the higher the A/B value, the more likely the chromatin is to be in an open state, and the more active the gene transcription is. ), and after mapping back to the slice, it was found that the spA/B value was also tissue-specific ( Figure 5C, Figure 5D).
  • the pixels of the liver area are extracted separately, combined with single-cell transcriptome data, to calculate the A/B components value of its marker gene, and annotate the main cell types in this area as Hepatocytes and Erythroid lineage.
  • SpatialHi-C can not only be a method to capture the three-dimensional conformation of chromatin with spatial resolution, but can also be used for joint analysis with other omics data, such as single-cell transcriptome, to study the three-dimensional conformation of chromatin from multiple dimensions.
  • omics data such as single-cell transcriptome
  • Table 14 shows the bioinformatics analysis results of the sequencing data obtained from the Spatial Hi-C experiment on mouse E13.5 day tissue sections.
  • raw reads the total number of reads obtained from sequencing
  • barcode the number of reads that are correctly connected to Barcode H and Barcode V
  • mapping the number of reads that are correctly mapped to the mouse reference genome
  • valid reads the total number of reads that contain chromatin three-dimensional conformation information ;trans: the number of reads containing interactions between different chromosomes; cis_short range contact( ⁇ 20kb): the number of reads containing short-range interactions within chromosomes; cis_long range contact(>20kb): the number of reads containing long-range interactions within chromosomes ; pix num: the number of all Pixels that can detect reads; mean cis long: the average number of long-distance interaction reads within each pixel chromosome.
  • Figure 6 shows the joint analysis of the A/B values of the pixels in the liver region in the mouse E13.5 day tissue sections and the single-cell transcriptome in the embodiment of the present invention.

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Abstract

Procédé de capture de conformation tridimensionnelle de la chromatine, comprenant les étapes suivantes : ligature de proximité de la chromatine in situ ; et marquage spécifique d'informations de localisation spatiale sur un produit de ligature de proximité, de sorte que la capture de conformation tridimensionnelle de la chromatine soit réalisée dans le cas d'une résolution spatiale conservée.
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