Method for constructing sequencing library and method based on enriched cell specific chromatin open sequencing technology

Abstract

The invention provides a construction method of an ATAC-seq library for cell specificity. The construction method comprises the following steps: fragmenting a genome of a suspension containing a cell nucleus or a single cell by using Tn5 transposase, and then fixing by using polyformaldehyde; then, specifically combining target nucleic acid by using paired probes, controlling the spacing, connecting the insert fragment and the skeleton to form a single chain ring, generating a long single chain of multiple repetitive units through rolling circle amplification, marking the repetitive units by using a third probe with a detectable signal, and sorting target cells based on the signal; and finally, carrying out decrosslinking on the sorted cells and carrying out PCR (Polymerase Chain Reaction) amplification by using a primer with a sequencing joint to obtain the sequencing library. The library constructed by the method can realize efficient enrichment of target cells in a pre-sequencing stage, and high-quality omics data of the target cells can be directionally obtained, so that open information of chromatin of a target cell type is captured.

Description

Technical Field

[0001] This application relates to the field of biotechnology, specifically to the field of single-cell sequencing, and more specifically to methods for constructing sequencing libraries and methods for cell-specific open sequencing based on enriched chromatin. Background Technology

[0002] The epigenetic state of chromatin is crucial for understanding gene expression and cellular state, encompassing dimensions such as chromatin openness, histone modifications, and DNA modifications. Among these, chromatin openness reflects the transcriptional regulatory state of cells under specific physiological or pathological conditions, playing a significant biological role in gene expression regulation, cell fate determination and differentiation, as well as stress responses and diseases.

[0003] To date, a series of sequencing technologies, including DNase-seq, FAIRE-seq, ATAC-seq, and MNase-seq, have been developed to capture information about chromatin openness. Among them, ATAC-seq has become the most widely used technology due to its low cell requirement and high sequencing quality. Its principle involves using engineered Tn5 transposase to "insert" DNA fragments with sequencing adapters into open regions of chromatin. Insertion occurs in regions where the DNA is not densely surrounded by nucleosomes; therefore, Tn5 preferentially inserts into open chromatin. After adapter ligation, PCR amplification and sequencing are performed, and finally, mapping analysis is performed on the reads to identify open regions.

[0004] However, the ATAC-seq technology itself provides limited information, only reflecting the arrangement of nucleosomes on chromatin and the binding of chromatin-binding proteins, but not the binding properties and aggregation of chromatin-binding proteins.

[0005] ACC-seq (Assay for Chromatin-bound Condensates by exploratory Sequencing), developed based on ATAC-seq, provides a new technical means to solve the above problems. ACC-seq is a chromatin openness detection method based on cross-linking agents and small molecule intervention, which is used to identify the interaction sites between protein condensates and chromatin across the entire genome.

[0006] With the development of sequencing technology, ATAC-seq can now achieve high throughput and capture chromatin open information at the single-cell level. However, this technology still has significant limitations; it cannot simultaneously satisfy cell type specificity and depth of capture. Even high-throughput single-cell sequencing technology captures cells randomly from tissues, making it difficult to target specific cell groups for analysis. ACC-seq also suffers from the problem of cell capture specificity, making it difficult to apply to tissues with high heterogeneity. Furthermore, ACC-seq technology itself requires a very high cell input (e.g., 100,000 cells), which greatly limits its application.

[0007] Therefore, current methods for open chromatin sequencing still need improvement. Summary of the Invention

[0008] The present invention aims to solve at least one of the technical problems existing in the prior art.

[0009] According to embodiments of this application, this application proposes a method for effectively sorting cellular units and further utilizes ATAC-seq and ACC-seq sequencing to efficiently obtain chromatin accessibility information. Specifically, this invention is mainly based on the following research idea: different types of cells have different gene expression levels and all specifically express certain RNA molecules. Even for many cell nuclei that have lost protein markers, they still contain many cell-specific RNA molecules. Therefore, the RNA fluorescence in situ hybridization (RNA FISH) technology according to embodiments of this application can be used to specifically label the target cell population, and the target cell population can be obtained by flow cytometry sorting for ATAC-seq and ACC-seq, thereby solving the problem of capturing cell population specificity by ATAC-seq and ACC-seq.

[0010] Therefore, in a first aspect of this application, a method for constructing a sequencing library is proposed, the sequencing library being used for cell-specific ATAC-seq chromatin open sequencing. The method includes: obtaining a suspension containing cell units, the cell units comprising at least one of a single cell nucleus and a single cell, the cell units containing a target nucleic acid sequence; using Tn5 transposase to fragment the genome of the cell units; immobilizing the fragmented cell units using polyformaldehyde solution; fluorescently labeling the immobilized cell units; sorting the cell samples based on the fluorescence to obtain desired cell units; decrosslinking the desired cell units; and performing PCR amplification on the decrosslinked cell units. R-amplification uses primers carrying sequencing adapters to obtain the sequencing library. The fluorescent labeling process includes: specifically binding a first probe and a second probe to the target nucleic acid sequence, wherein the binding site of the first probe and the binding site of the second probe are spaced no more than a predetermined length apart; providing an insert, a backbone fragment, and a ligase, wherein the nucleic acid sequences of the insert and the backbone fragment are configured to be ligated into a single-stranded loop when the first and second probes specifically bind to the target nucleic acid sequence; performing rolling circle replication on the single-stranded loop to obtain a long single-stranded molecule containing multiple repeating units; binding a third probe to the repeating units, the third probe carrying a detectable signal; and sorting the cell units based on the detectable signal.

[0011] According to embodiments of this application, this application provides a means for enriching various cellular units, such as single cells and single cell nuclei, in single-cell sequencing technology. Therefore, according to embodiments of this application, this application proposes a single-cell sequencing technology with cell type-specific enrichment capabilities. By employing this technology, efficient enrichment of target cells can be achieved in the pre-sequencing stage, ensuring that subsequent single-cell sequencing can target high-quality data from the target cells, significantly improving research efficiency and data output value. This technological breakthrough will provide strong technical support for cutting-edge fields such as rare cell research and disease mechanism analysis. Furthermore, it provides an effective means to obtain chromatin openness information through sequencing.

[0012] In a second aspect of this application, a method for constructing a sequencing library is proposed. According to an embodiment of this application, the sequencing library is used for cell-specific ACC-seq open chromatin sequencing. The method includes: obtaining a suspension containing cell units, wherein the cell units include at least one of a single cell nucleus and a single cell, and the cell units contain a target nucleic acid sequence; dividing the suspension into a Fresh group, a formaldehyde-fixed group, and a 1,6-hexanediol-treated group, and performing respective fragmentation treatments on the Fresh group, the formaldehyde-fixed group, and the 1,6-hexanediol-treated group to fragment the chromatin using Tn5 transposase; immobilizing the fragmented cell units using polyoxymethylene solution; and fluorescently reacting the immobilized cell units with... The process includes: optical labeling; sorting the cell samples based on the fluorescence to obtain desired cell units; decrosslinking the desired cell units; and performing PCR amplification on the decrosslinked cell units using primers carrying sequencing adapters to obtain the sequencing library, which includes a Fresh sequencing library, a formaldehyde-fixed sequencing library, and a 1,6-hexanediol-treated sequencing library. The fluorescent labeling process includes: specifically binding a first probe and a second probe to the target nucleic acid sequence, wherein the binding site of the first probe and the binding site of the second probe are spaced apart by a predetermined length; and providing an insert fragment, a backbone fragment, and a ligase. The nucleic acid sequences of the insert fragment and the backbone fragment are configured such that, when the first and second probes specifically bind to the target nucleic acid sequence, the insert fragment and the backbone fragment can be linked into a single-stranded loop; the single-stranded loop is subjected to rolling circle replication to obtain a long single-stranded molecule containing multiple repeating units; a third probe is made to bind to the repeating units, the third probe carrying a detectable signal; and the cell units are sorted based on the detectable signal, wherein the fragmentation treatment of the Fresh group is performed by reacting the cell units of the Fresh group with a reaction mixture containing Tn5 transposase, and the fragmentation treatment of the formaldehyde-fixed group includes: The cell units in the formaldehyde-fixed group are fixed with formaldehyde using a polyoxymethylene solution; and the formaldehyde-fixed cell units are reacted with a reaction mixture containing Tn5 transposase to break down the chromatin of the cell units. The breakdown process in the 1,6-hexanediol-treated group includes: treating the cell units in the 1,6-hexanediol-treated group to disrupt chromatin aggregates and release chromatin regions encapsulated by aggregates; fixing the treated cell units with formaldehyde using a polyoxymethylene solution; and reacting the formaldehyde-fixed cell units with a reaction mixture containing Tn5 transposase to break down the chromatin of the cell units.

[0013] This method enables the efficient construction of sequencing libraries suitable for ACC-seq. According to embodiments of this application, ACC-seq is a chromatin accessibility detection method based on cross-linking agents and small molecule interventions, used to identify interaction sites between protein condensates and chromatin across the entire genome. This method treats cells with cross-linking agents to modulate the physical state of chromatin-protein condensate binding, affecting the integration efficiency and localization specificity of transposases in this region. The aforementioned cross-linking treatment can inhibit the binding of transposases in protein condensate-rich regions, reflecting a decrease in chromatin accessibility. Furthermore, by treating cells with specific small molecule compounds, the phase separation state of protein condensates can be selectively disrupted. This intervention can significantly alleviate the inhibitory effect of cross-linking agent fixation on chromatin accessibility at condensate binding sites, thereby restoring transposase activity in this region. By comparing the differences in chromatin accessibility under different treatment conditions (i.e., the Fresh group, cross-linking agent treatment group, and small molecule intervention group in this application), combined with high-throughput sequencing technology, chromatin regions associated with protein condensate binding can be systematically identified at the whole-genome level. Therefore, the method according to the embodiments of this application can be widely used to study the relationship between chromatin phase separation structure and gene regulation, and can be used for the identification and screening of functional condensates.

[0014] According to an embodiment of this application, in a third aspect of this application, a method based on enriched cell-specific chromatin openness sequencing technology is proposed, comprising: constructing a sequencing library for a sample to be tested according to the method described in the first or second aspect; sequencing the sequencing library; and analyzing the sequencing results to obtain chromatin openness information of the sample to be tested.

[0015] Therefore, according to embodiments of this application, specific cell populations can be captured in tissues via RNA FISH, and combined with chromatin accessibility information obtained by ATAC-seq. According to embodiments of this application, a strategy of treating with Tn5 transposase followed by fixation is used to avoid the impact of fixed cells on the efficiency of Tn5 transposase treatment, significantly improving the data quality of ATAC-seq. According to embodiments of this application, ACC-seq can be performed in highly heterogeneous tissues to obtain information on the interaction sites between protein condensates and chromatin of specific cell populations. Furthermore, according to embodiments of this application, this technology requires a small number of cells, achieves high sequencing quality, and while ATAC-seq can acquire high-quality data at the single-cell level, ACC-seq can acquire high-quality data at approximately 3000 cells. Attached Figure Description

[0016] Figure 1This diagram illustrates the principle of chromatin openness analysis technology based on RNA FISH enrichment according to embodiments of this application. In the diagram, a. represents the experimental principle of enriching and sorting target cell types for chromatin openness analysis, including the fragmentation of single cells or nuclei from tissues by Tn5 transposase and the addition of adapters, followed by formaldehyde fixation, labeling of target cell types using RNA FISH technology, enrichment of target cells through flow cytometry, and subsequent ATAC-seq library construction. b. represents the principle and flowchart of RNA FISH technology, including two probes (left and right) forming a pair, hybridizing to adjacent positions on the same RNA molecule. The backbone DNA and insert DNA further bind to the probe molecule ends, and under the action of T4 ligase, the backbone DNA and insert DNA are ligated into a single-stranded circular DNA. Phi29 DNA polymerase initiates a rollover replication reaction to form long single-stranded DNA, and finally, the fluorescent probe hybridizes to the long single-stranded DNA for imaging.

[0017] Figure 2 The figure shows RNA FISH staining and flow cytometry sorting of cell lines and mouse hippocampus according to embodiments of this application. In this figure, a. Staining results of mouse cell line Tyr gene probe RNA FISH; the left panel shows B16 cells with high Tyr mRNA expression, and the right panel shows 4T1 cells with low Tyr mRNA expression. b. Flow cytometry analysis results of human CLU gene staining in HeLa (human) and NIH / 3T3 (mouse) cell nuclei; scale bar is 10 μm. c. Mouse hippocampal staining results; P1 are Tubb3-positive nuclei. P2 are Tubb3-positive and Arc-positive nuclei, and P3 are Tubb3-positive and Arc-negative nuclei. d. qPCR quantification of gene expression levels in nuclei sorted from different phyla in figure c. Flow cytometry sorted 100 nuclei for reverse transcription and qPCR quantification; the Y-axis represents the absolute Cq value of gene expression.

[0018] Figure 3 The quality of RNA FISH-enriched nuclear ATAC-seq data according to embodiments of this application is shown. In this figure, a. IGV screenshots of signal peaks after alignment of single-nucleus ATAC-seq data with the genome. b. Statistical graph of the fraction of sequencing reads in signal peaks (FRiP) in bulk (100 nuclei) and single-nucleus ATAC-seq data. c. Changes in chromatin accessibility at the Arc gene location in RNA FISH-enriched mouse hippocampal Arc-positive neurons.

[0019] Figure 4A schematic diagram of the ACC-seq technology according to an embodiment of this application is shown. In this diagram, the method utilizes paraformaldehyde as a cross-linking agent to detect chromatin-associated aggregate structures. In the native state of cells, the Tn5 transposase can enter chromatin, including regions occupied by aggregates. However, after paraformaldehyde fixation, the cross-linked aggregates restrict Tn5's cleavage and insertion of DNA, thereby inhibiting transcriptase reactions in that region. If cells are treated with 1,6-hexanediol (1,6-Hex) before fixation, these liquid-liquid phase separation aggregates can be dissolved, releasing the chromatin structures within, thereby enhancing Tn5's entry and tagging response in the relevant regions. Through subsequent high-throughput sequencing and chromatin accessibility analysis, different regions in the genome can be divided into two categories: hexanediol-sensitive regions: sensitive to 1,6-Hex treatment, indicating that their chromatin accessibility is significantly affected by aggregates; and hexanediol-insensitive regions: insensitive to 1,6-Hex treatment, with minimal impact from aggregates.

[0020] Figure 5 The image shows a screenshot of the IGV signal at the Junb gene location in mouse hippocampal neurons according to an embodiment of this application via ACC-seq. In this figure, the cell population consists of mouse hippocampal neurons labeled with the Tubb3 probe, and ACC-seq library construction and analysis were performed. The red box marks the chromatin region near the Junb gene that is sensitive to 1,6-hexanediol. Detailed Implementation

[0021] The embodiments of the present invention are described in detail below. The embodiments described below are exemplary and intended to explain the present invention, and should not be construed as limiting the present invention.

[0022] According to embodiments of this application, this application proposes a method for effectively sorting cellular units and further utilizes ATAC-seq and ACC-seq sequencing to efficiently obtain chromatin accessibility information. Specifically, this invention is mainly based on the following research idea: different types of cells have different gene expression levels and all specifically express certain RNA molecules. Even for many cell nuclei that have lost protein markers, they still contain many cell-specific RNA molecules. Therefore, the RNA fluorescence in situ hybridization (RNA FISH) technology according to embodiments of this application can be used to specifically label the target cell population, and the target cell population can be obtained by flow cytometry sorting for ATAC-seq and ACC-seq, thereby solving the problem of capturing cell population specificity by ATAC-seq and ACC-seq.

[0023] According to embodiments of this application, a method capable of effectively sorting cell units is employed, thereby providing a means to enrich various cell units, such as single cells and single cell nuclei, for single-cell sequencing technology. Thus, according to embodiments of this application, a single-cell sequencing technology with cell type-specific enrichment function is proposed. By employing this technology, efficient enrichment of target cells can be achieved in the pre-sequencing stage, ensuring that subsequent single-cell sequencing can target high-quality data from the target cells, significantly improving research efficiency and data output value. This technological breakthrough will provide strong technical support for cutting-edge fields such as rare cell research and disease mechanism analysis.

[0024] Therefore, in the first aspect of this application, a method for constructing a sequencing library is proposed, referring to... Figure 1 The sequencing library is used for ATAC-seq open chromatin sequencing. The method includes: obtaining a suspension containing cell units, wherein the cell units include at least one of a single cell nucleus and a single cell, and the cell units contain a target nucleic acid sequence; using Tn5 transposase to fragment the genome of the cell units; immobilizing the fragmented cell units using polyformaldehyde solution; fluorescently labeling the immobilized cell units; sorting the cell samples based on the fluorescence to obtain desired cell units; decrosslinking the desired cell units; and performing PCR amplification on the decrosslinked cell units using primers carrying sequencing adapters to obtain... The sequencing library, wherein the fluorescent labeling treatment includes: specifically binding a first probe and a second probe to the target nucleic acid sequence, wherein the binding site of the first probe and the binding site of the second probe are spaced apart by no more than a predetermined length; providing an insert, a backbone fragment, and a ligase, wherein the nucleic acid sequences of the insert and the backbone fragment are configured to be ligated into a single-stranded loop when the first and second probes specifically bind to the target nucleic acid sequence; performing rolling circle replication on the single-stranded loop to obtain a long single-stranded molecule, the long single-stranded molecule comprising multiple repeating units; binding a third probe to the repeating units, the third probe carrying a detectable signal; and sorting the cell units based on the detectable signal.

[0025] According to embodiments of this application, this application can enrich various cell units, such as single cells and single cell nuclei, for single-cell sequencing technology. Therefore, according to embodiments of this application, this application proposes a single-cell sequencing technology with cell type-specific enrichment function. By employing this technology, efficient enrichment of target cells can be achieved in the pre-sequencing stage, thereby ensuring that subsequent single-cell sequencing can directionally acquire high-quality data from target cells, significantly improving research efficiency and data output value. This technological breakthrough will provide strong technical support for cutting-edge fields such as rare cell research and disease mechanism analysis. Furthermore, it provides an effective means to obtain chromatin openness information through sequencing.

[0026] In a second aspect, this application proposes a method for constructing a sequencing library, characterized in that the sequencing library is used for ACC-seq open chromatin sequencing, the method comprising: obtaining a suspension containing cell units, the cell units including at least one of a single cell nucleus and a single cell, the cell units containing a target nucleic acid sequence; dividing the suspension into a Fresh group, a formaldehyde-fixed group, and a 1,6-hexanediol-treated group, and performing respective fragmentation treatments on the Fresh group, the formaldehyde-fixed group, and the 1,6-hexanediol-treated group to fragment the chromatin using Tn5 transposase; immobilizing the fragmented cell units using polyoxymethylene solution; and immobilizing the immobilized cell units using polyoxymethylene solution. The cell units treated with fluorescein are subjected to fluorescent labeling; based on the fluorescence, the cell samples are sorted to obtain the desired cell units; the desired cell units are subjected to decrosslinking treatment; the decrosslinked cell units are subjected to PCR amplification using primers carrying sequencing adapters to obtain the sequencing library, which includes a Fresh sequencing library, a formaldehyde-fixed sequencing library, and a 1,6-hexanediol-treated sequencing library. The fluorescent labeling treatment includes: specifically binding a first probe and a second probe to the target nucleic acid sequence, wherein the binding site of the first probe and the binding site of the second probe are spaced no more than a predetermined length apart; and providing an insert fragment and a backbone. The method involves a segment and a ligase, wherein the nucleic acid sequences of the insert fragment and the backbone fragment are configured such that, when the first and second probes specifically bind to the target nucleic acid sequence, the insert fragment and the backbone fragment can be ligated into a single-stranded loop; the single-stranded loop is subjected to rolling circle replication to obtain a long single-stranded molecule containing multiple repeating units; a third probe is made to bind to the repeating units, the third probe carrying a detectable signal; and the cell units are sorted based on the detectable signal, wherein the fragmentation treatment of the Fresh group is performed by reacting the cell units of the Fresh group with a reaction mixture containing Tn5 transposase, and the fragmentation treatment of the formaldehyde-fixed group is performed... The method includes: fixing the cell units in the formaldehyde-fixed group with formaldehyde using a polyoxymethylene solution; and reacting the formaldehyde-fixed cell units with a reaction mixture containing Tn5 transposase to break down the chromatin of the cell units. The breakdown process in the 1,6-hexanediol-treated group includes: treating the cell units in the 1,6-hexanediol-treated group to disrupt chromatin aggregates and release chromatin regions encapsulated by the aggregates; fixing the treated cell units with formaldehyde using a polyoxymethylene solution; and reacting the formaldehyde-fixed cell units with a reaction mixture containing Tn5 transposase to break down the chromatin of the cell units.

[0027] This method enables the efficient construction of sequencing libraries suitable for ACC-seq. According to embodiments of this application, ACC-seq is a chromatin accessibility detection method based on cross-linking agents and small molecule interventions, used to identify interaction sites between protein condensates and chromatin across the entire genome. This method treats cells with cross-linking agents to modulate the physical state of chromatin-protein condensate binding, affecting the integration efficiency and localization specificity of transposases in this region. The aforementioned cross-linking treatment can inhibit the binding of transposases in protein condensate-rich regions, reflecting a decrease in chromatin accessibility. Furthermore, by treating cells with specific small molecule compounds, the phase-separation state of protein condensates can be selectively disrupted. This intervention significantly alleviates the inhibitory effect of cross-linking agent fixation on chromatin accessibility at condensate binding sites, thereby restoring transposase activity in this region. By comparing the differences in chromatin accessibility under different treatment conditions (cross-linking agent treatment, small molecule intervention treatment, combined treatment, and control group), combined with high-throughput sequencing technology, chromatin regions associated with protein condensate binding can be systematically identified at the whole-genome level. Therefore, the method according to the embodiments of this application can be widely used to study the relationship between chromatin phase separation structure and gene regulation, and can be used for the identification and screening of functional condensates.

[0028] In a third aspect of this application, a method based on enriched cell-specific chromatin openness sequencing technology is proposed, comprising: constructing a sequencing library for a sample to be tested according to the method described in the first or second aspect; sequencing the sequencing library; and analyzing the sequencing results to obtain chromatin openness information of the sample to be tested.

[0029] Therefore, according to embodiments of this application, specific cell populations can be captured in tissues via RNA FISH, and combined with chromatin accessibility information obtained by ATAC-seq. According to embodiments of this application, a strategy of treating with Tn5 transposase followed by fixation is used to avoid the impact of fixed cells on the efficiency of Tn5 transposase treatment, significantly improving the data quality of ATAC-seq. According to embodiments of this application, ACC-seq can be performed in highly heterogeneous tissues to obtain information on the interaction sites between protein condensates and chromatin of specific cell populations. Furthermore, according to embodiments of this application, this technology requires a small number of cells, achieves high sequencing quality, and while ATAC-seq can acquire high-quality data at the single-cell level, ACC-seq can acquire high-quality data at approximately 3000 cells.

[0030] refer to Figure 1 According to the principle and flowchart of PLAYR in the embodiments of this application, the method mainly includes the following steps:

[0031] Step 1: DNA probes (probe 1 and probe 2) hybridize with RNA molecules; Step 2: Backbone DNA and insert DNA hybridize with RNA probes; Step 3: T4 DNA ligase ligates backbone DNA and insert DNA to form circular DNA molecules; Step 4: Rolling circle replication reaction, probe 2 uses the circular DNA molecule as a template to extend into long single-stranded DNA under the action of DNA polymerase; Step 5: Fluorescent probe molecules hybridize with long single-stranded DNA, generating a fluorescent signal.

[0032] Specifically, according to embodiments of this application, the method includes: specifically binding a first probe and a second probe to the target nucleic acid sequence, wherein the binding site of the first probe and the binding site of the second probe are spaced apart by no more than a predetermined length; providing an insert fragment, a backbone fragment, and a ligase, wherein the nucleic acid sequences of the insert fragment and the backbone fragment are configured such that, when the first probe and the second probe specifically bind to the target nucleic acid sequence, the insert fragment and the backbone fragment can be linked into a single-stranded loop; performing rolling circle replication on the single-stranded loop to obtain a long single-stranded molecule, the long single-stranded molecule comprising multiple repeating units; binding a third probe to the repeating units, the third probe carrying a detectable signal; and sorting the cell units based on the detectable signal.

[0033] According to embodiments of this application, by employing this method of sorting cell units, the detectable signal carried by the third probe can be used to identify cell units expressing the target gene, such as single cells or single nuclei. Furthermore, based on the difference in the detectable signal, cell units with different gene expression levels, such as single cells or single nuclei, can be distinguished. This allows for efficient selection of target cell populations before sequencing. Specifically, according to embodiments of this application, probes targeting different genes can be designed to label cells dissociated from tissues or extract cell nuclei. Flow cytometry sorting technology, based on the combination and intensity of fluorescence signals, can sort and capture single cells or single nuclei of the target cell population for omics library construction and sequencing analysis, thereby achieving enriched sequencing of the target cell population. This effectively addresses the random capture limitations of current single-cell sequencing technology, enabling the effective acquisition of rare cell types (such as neural stem cells, which account for only 0.1% of the adult hippocampus) and the effective capture of dynamic cells (such as embryonic neural stem cells), which are currently difficult to capture effectively.

[0034] As used herein, the term "rare cells" refers to a subpopulation of cells that constitutes less than 1% of the total cell population in a biological sample, such as approximately one in a thousand. These subpopulations typically possess unique biological functions or phenotypic characteristics that are difficult to effectively isolate and identify using conventional single-cell techniques. As an example, the technique of this invention can sort and enrich rare cells including adult neural stem cells, circulating tumor cells, hematopoietic stem cells, and tumor-initiating cells. Adult neural stem cells are distributed in the dentate gyrus of the hippocampus and the subependymal region of the lateral ventricle, accounting for approximately 0.1% of hippocampal cells. Traditional flow cytometry sorting relies on membrane markers (such as Prom1), but these markers are lost during the extraction of tissue cell nuclei. The technique of this invention, however, achieves high enrichment efficiency by labeling Tubb3 / Arc mRNA.

[0035] According to embodiments of this application, the cellular unit includes at least one of a single cell, a single cell nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosome, and exosome. According to embodiments of this application, the inventors have discovered that the technical solution of this application is applicable not only to single cells but also to subcellular organelles and other cellular units, such as the cell nucleus, mitochondria, endoplasmic reticulum, Golgi apparatus, lysosome, and exosome. These cellular units all contain genetic material and can be labeled with specific targets as needed, thereby achieving effective sorting. Furthermore, for certain scenarios where it is difficult to separate single cells, the chromatin openness of these cells can be effectively analyzed by separating subcellular organelles, especially the single cell nucleus. For example, in complex neural or tumor tissues, because the intercellular connections in these tissues are very tight, it is difficult to dissociate the tissue to obtain a single-cell suspension without affecting cell integrity, or the dissociation process is biased towards different cell types, resulting in sequencing results that cannot reflect the true situation in vivo. For these tissues, according to embodiments of this application, single-cell nuclear sequencing can be used to characterize the gene expression of single cells and can effectively perform sorting. However, existing antibody labeling methods cannot achieve effective sorting because biomarkers on the cell membrane surface are lost during the extraction of cell nuclei from tissue. Therefore, these markers can no longer be used to label specific cell types for cell enrichment, and the number of biomarkers on the nuclear membrane surface that have been identified is very limited.

[0036] It should be noted that there are no particular restrictions on the methods for obtaining suspensions containing cellular units; conventional methods can be used. For example, a suspension of single cells or cell nuclei can be obtained by enzymatically digesting biological tissues.

[0037] According to embodiments of this application, the target nucleic acid sequence is DNA or RNA, optionally, the target nucleic acid sequence is mRNA. According to embodiments of this application, the type of target nucleic acid sequence that can be used is not particularly limited; it can be either DNA or RNA, and can be selected according to the specific cell unit of interest being analyzed. According to embodiments of this application, RNA, especially mRNA, is preferably used as the target nucleic acid sequence. This allows for the use of these target nucleic acid sequences as markers based on the differences in expression levels of RNA, especially mRNA, in different cells, achieving specific cell sorting. According to embodiments of this application, the inventors have discovered that because the method of this application has a short hybridization processing time and a high signal amplification factor, it can be effectively applied to RNA, especially mRNA, as the target sequence, further improving the specific labeling and sorting of cell units, especially for single cells or single cell nuclei. Specifically, according to embodiments of this application, in situ fluorescent labeling of cell units can be completed within 9 hours, while traditional labeling techniques require overnight reactions or even longer. Therefore, the technical solution of this invention can rapidly and efficiently label RNA molecules, especially mRNA, while avoiding the degradation of these substances.

[0038] According to embodiments of this application, by employing a first probe, a second probe, an insert fragment, and a backbone fragment, the insert fragment and backbone fragment can be ligated into a single-stranded DNA circular under the action of a ligase. Specifically, by setting the sequences of the first probe, the second probe, the insert fragment, and the backbone fragment, it is suitable for forming a single-stranded DNA circular using PLAYR (proximity ligation assay for RNA). As an example, see [reference]. Figure 2The first and second probes specifically bind to target molecules, such as mRNA molecules. Since the binding site of the first probe and the binding site of the second probe are separated by a predetermined length, for example, no more than 10 bp, or even no more than 5 bp, preferably 3-5 bp, circular ligation can be facilitated by bringing the insert fragment and backbone fragment that can match the first and second probes closer together. As an example, according to an embodiment of this application, the lengths of the first and second probes are independently 15-25 bp, preferably approximately 20 bp. According to an embodiment of this application, the Tm values ​​of the first and second probes are independently 58-60 degrees Celsius. According to an embodiment of this application, the GC content of the first and second probes is independently 30-70%. According to an embodiment of this application, the insert fragment can simultaneously match the first and second probes, and the two ends of the backbone fragment can bind to the first and second probes respectively, thereby bringing the insert fragment and backbone fragment closer together under the action of the first and second probes, thus achieving circular ligation. As an example, paired DNA probes (probe 1 and probe 2) are designed based on the RNA sequence. When probe 1 and probe 2 bind to adjacent sites on the RNA, the inserted DNA and backbone DNA can bind to the probes and, under the action of T4 DNA ligase, the inserted DNA and backbone DNA are linked into a single-stranded DNA loop.

[0039] According to embodiments of this application, the ligase is T4 DNA ligase, and the rolling circle replication uses Phi29 DNA polymerase. According to embodiments of this application, single-stranded DNA loops can undergo rolling circle replication under the action of Phi29 DNA polymerase. For example, a second probe can be used as a primer to initiate the rolling circle reaction, generating a long single-stranded molecule. This long single-stranded molecule includes multiple repetitions of the single-stranded DNA loop sequence, thus containing multiple repeating units. By using a third probe capable of binding to the repeating units, a detectable signal can be labeled, thereby further enabling the sorting of cell units based on the detectable signal. Specifically, rolling circle replication can be initiated using either the first or second probe as a primer. The third probe can be designed based on a portion or the entire length of the insert fragment and backbone fragment. For example, according to one embodiment of this application, a sequence containing the insert fragment can be used as the third probe. According to specific embodiments of this application, by designing a probe identical to the insert fragment and modified with fluorescence (the third probe), dozens or even hundreds of binding sites can be found on the long single strand, thereby achieving enrichment of the fluorescent signal near the target RNA. Different insertion fragments with different sequences can be designed to distinguish different target RNAs, thus enabling the simultaneous labeling of multiple RNA molecules in the same cell. According to embodiments of this application, the detectable signal is a fluorescence signal. According to embodiments of this application, the sorting is performed using flow cytometry.

[0040] According to embodiments of this application, the cell unit contains multiple different target nucleic acid sequences, each independently assigned the same or different detectable signals. According to embodiments of this application, the cell unit contains at least five different target nucleic acid sequences, for example, eight different target nucleic acid sequences. According to embodiments of this application, the multiple different target nucleic acid sequences originate from the same gene. According to embodiments of this application, there are no overlapping regions between the multiple different target nucleic acid sequences. Therefore, further amplification of detectable signals, such as fluorescence signals, can be achieved, improving sorting efficiency.

[0041] According to embodiments of this application, the cell units are immobilized before the first and second probes specifically bind to the target nucleic acid sequence. According to embodiments of this application, the immobilization is performed using polyoxymethylene (POM) (the terms "formaldehyde fixation" and "POM fixation" are used interchangeably herein). According to embodiments of this application, the immobilization is performed using a 37% PFA solution. This preserves the interactions between intracellular molecules, thereby preventing cell death, damage, and degradation. To address the impact of PFA fixation on RNA-seq sequencing quality, cells sorted by flow cytometry or cells sorted into a solution containing proteinase K and SDS (sodium dodecyl sulfate) can be decrosslinked at high temperatures to remove protein molecules crosslinked to mRNA molecules before library construction and sequencing. According to embodiments of this application, the inventors unexpectedly discovered that, for both single cells and cell nuclei, the proportion of sequencing fragments aligned to the genome in the decrosslinked sequencing samples did not significantly change compared to fresh cells. Furthermore, according to the embodiments of this application, the inventors surprisingly discovered that high-quality sequencing libraries can still be obtained from cells and nuclei that have undergone fixation treatment, especially information such as high-quality chromatin openness and gene expression levels.

[0042] According to an embodiment of this application, the nucleic acid molecule is RNA. According to an embodiment of this application, the desired cell units are subjected to decrosslinking treatment before constructing the sequencing library. According to an embodiment of this application, the decrosslinking treatment is performed by high-temperature treatment using a solution containing proteinase K and sodium dodecyl sulfate.

[0043] Therefore, according to the embodiments of this application, the present invention, by employing in situ fluorescently labeled RNA FISH technology, can label cells and nuclei expressing the target gene using fluorescent signals. Furthermore, flow cytometry can be used to distinguish cells and nuclei with different gene expression levels. Additionally, fixed cells and nuclei can still provide high-quality information on chromatin openness, gene expression levels, and other relevant data. According to the embodiments of this application, target cell populations are efficiently selected before sequencing. RNA FISH probes targeting different genes are designed to label cells dissociated from tissues or extract cell nuclei. Flow cytometry is then used to sort and capture single cells or single nuclei of the target cell population based on the combination and intensity of RNA FISH fluorescence signals. Omics library construction and sequencing analysis are then performed, thereby achieving enriched sequencing of the target cell population. Furthermore, according to the embodiments of this application, highly efficient RNA FISH can be achieved in single-cell and single-nucleus suspensions dissociated from tissue. This RNA FISH technology can be completed in just 8-9 hours, while traditional techniques require overnight reactions or even longer. This technology can screen cells or nuclei with different gene expression levels by the fluorescence intensity of RNA FISH. This technology can simultaneously label multiple RNA molecules and can sort corresponding cell groups by combining fluorescence signals.

[0044] In addition, the inventors have discovered that the in situ fluorescent labeling technology of this application is applicable to RNA molecules with different expression levels. It only requires the design of different types of probes based on the sequence of the RNA molecule. Moreover, the technology has low requirements for probe molecule synthesis and only requires ordinary desalting and purification strategies. The technology has low cost and is easy to promote and use.

[0045] Therefore, according to the embodiments of this application, such as Figure 1 As shown in Figure a, single cells or single cell nuclei isolated from tissue are first subjected to Tn5 transposase to break down the genome in vitro, followed immediately by formaldehyde fixation of the nuclei to prevent DNA fragment leakage. Then, RNA FISH technology is used to label the target cell population based on nucleus-specific RNA molecules. The target cells are then obtained through flow cytometry sorting and decrosslinking treatment, followed by ATAC-seq library construction and amplification. The principle of RNA FISH technology is as follows: Figure 1As shown in b, this technology is based on PLAYR (Proximity Ligation Assay for RNA). It involves designing multiple pairs of adjacent probe molecules that bind to the target RNA. The probe molecules contain fixed sequences at their ends, providing binding sites for the backbone DNA molecule and the insert DNA molecule. When the backbone DNA molecule and the insert DNA molecule bind to the probe, T4 DNA ligase ligates them into a single-stranded circular DNA molecule. This circular DNA molecule is then extended into a long single-stranded DNA molecule by phi29 DNA polymerase. The sequence of this long single-stranded DNA molecule is a continuous repetition of the circular DNA molecule sequence. Therefore, by designing a fluorescent probe with the same sequence as the insert DNA molecule, enrichment of the fluorescent probe on the target RNA molecule can be achieved.

[0046] According to embodiments of this application, the inventors verified the specificity of RNA FISH staining in cell lines, such as... Figure 2 As shown in Figure a, the RNA FISH labeling and sorting technology according to the embodiments of this application can screen for target cell populations on a flow cytometer. Furthermore, according to the embodiments of this application, the inventors also tested this technology in highly heterogeneous tissues, and the inventors further demonstrated using mouse hippocampal tissue that this technology can be efficiently applied to the sorting of target cell populations in complex tissues.

[0047] According to embodiments of this application, for capturing chromatin openness, the inventors typically use ATAC-seq. This technique uses Tn5 transposase to break down DNA in open regions of the genome. However, paraformaldehyde fixation of cells or nuclei affects the efficiency of Tn5 transposase penetration and access to chromatin. Furthermore, the cross-linking of DNA molecules with formaldehyde molecules and DNA-binding proteins also affects the efficiency of transposase in breaking down the genome, significantly reducing the data quality of ATAC-seq. To address the impact of PFA fixation on ATAC-seq, the inventors employ a technique whereby Tn5 transposase is used to break down the chromatin in all cell nuclei before paraformaldehyde fixation. Immediately after this treatment, paraformaldehyde is used to fix the cell nuclei, immobilizing the fragmented DNA within the nuclei. Subsequently, RNA FISH is performed to label the target cell population. The sorted cell nuclei are then decross-linked, releasing the fragmented DNA, which will be used for subsequent library construction in ATAC-seq. Figure 3 As shown in figure a, single-nucleus ATAC-seq also exhibits a high signal-to-noise ratio and reproducibility, with clear signal peaks observed in regions such as gene promoters. Although the inventors can obtain high-quality single-nucleus ATAC-seq data, a large number of nuclei (100 nuclei) can provide more effective information, and the proportion of sequencing reads falling on ATAC-seq signal peaks (FRiP) is significantly higher than that of single-nucleus data. Figure 3 As shown in b. Therefore, a large amount of ATAC-seq data from cell nuclei will more effectively help the inventors study changes in chromatin openness in specific biological processes. Furthermore, for the activated Arc-positive neurons mentioned above, the chromatin openness in the promoter region of the Arc gene is significantly increased compared to unactivated neurons, reflecting the high expression of the Arc gene in Arc-positive neurons, further demonstrating the accuracy of the inventors' enrichment sequencing technology, such as... Figure 3 As shown in c.

[0048] Furthermore, according to embodiments of this application, the inventors tested the ACC-seq technology based on RNA FISH sorting, and the detailed technical principles are as follows: Figure 4 The dissociated single-cell or single-nuclear suspensions were divided into three groups: the first group (Fresh group) involved cells first undergoing Tn5 transposase fragmentation followed by formaldehyde fixation to obtain chromatin openness information in the original cellular state; the second group (Formaldehyde-fixed group) involved cells first undergoing formaldehyde fixation to immobilize condensates on chromatin, followed by Tn5 transposase fragmentation; and the third group (1,6-hexanediol-treated group) involved cells first undergoing 1,6-hexanediol treatment to disrupt condensates on the chromatin, releasing the chromatin regions previously enclosed by condensates, followed by formaldehyde fixation and then Tn5 transposase fragmentation of the chromatin. All three groups of cells were then subjected to parallel RNA FISH staining to label and sort the target cell populations, thereby achieving ACC-seq of the target cell types at the tissue level. After sequencing, the differences in chromatin openness signals among the three groups of cells were calculated using bioinformatics to identify 1,6-hexanediol-sensitive condensate-covered regions and 1,6-hexanediol-insensitive regions on the chromatin. According to an embodiment of this application, the inventors used RNA FISH to sort hippocampal neurons in a resting state of mice using a Tubb3 gene probe for ACC-seq. RNA FISH helped the inventors obtain target neurons from highly heterogeneous tissues, improving cell purity. The inventors obtained high-quality ACC-seq data using only about 3000 cells. Figure 5 As shown, a 1,6-hexanediol-sensitive signal peak is observed at the Junb gene location. Junb is an immediate early gene that can respond to neuronal stimulation and mediate neuronal function.

[0049] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention. The present invention will be described in detail below with reference to specific embodiments, which are used to understand rather than limit the present invention.

[0050] General methods

[0051] I. RNA FISH Probe Design Principles (First Probe and Second Probe)

[0052] 1. Download the RNA sequence of the target gene from the NCBI database and save it in FASTA format. If the RNA produced by gene transcription has multiple alternative splices, take the overlapping portions of these alternative splices.

[0053] 2. Using the R online editor, import the saved FASTA file into Primer3, design a binding sequence complementary to the target gene RNA, and design probe parameters based on the optimized signal amplification RNA FISH principle of this technology:

[0054] (1) The length of each of the two adjacent probes is about 20 bp. The binding sites on the RNA are 3-5 bp apart. Therefore, the total length of a pair of probes binding on the RNA is 42-45 bp.

[0055] (2) The Tm value of each probe is 58℃-60℃, and the GC content is 30%-70%.

[0056] 3. The probe sequences designed from Primer3 are placed into BLASTN to test their specificity. Sequence alignment reveals the non-specific binding sites that may occur in the genome and transcriptome of the corresponding species. Generally, if a probe exhibits non-specific binding beyond the target sequence of more than 30 bp, it is considered to have low specificity and is excluded from the list of possible probe sequences.

[0057] 4. Optionally, to avoid the probes themselves forming complex hairpin structures that could affect hybridization between probes, the probe sequences generated in the above steps can be imported into nupack to predict their molecular structures. By calculating the probability of forming secondary structures, probe sequences with fewer secondary structures can be further screened out.

[0058] 5. To label genes with moderate expression levels, it is necessary to design about 8 pairs of probes. Mark the binding sites of the probes selected in the above steps on the RNA, and then select 8 pairs of probes whose binding sites do not overlap.

[0059] Finally, the designed probe sequence was sent to the bioengineering synthesis department for synthesis, purified by desalting, and stored as a powder.

[0060] II. Cellular and Nuclear RNA FISH

[0061] RNA labeling was performed on deionized single-cell suspensions or extracted tissue single-cell nuclear suspensions, following the same cell staining procedures as for nuclei. The following is an example of the specific steps for nuclear RNA FISH:

[0062] 1. Add 37% polyoxymethylene solution to the cell nucleus suspension to achieve a final concentration of 1.6%, and then fix on ice for 10 minutes.

[0063] 2. Add Tris-HCl to a final concentration of 166.7 mM at pH 7.5 and incubate at room temperature for 10 minutes to bind unreacted paraformaldehyde and terminate fixation.

[0064] 3. After termination, add Triton X-100 to the solution to a final concentration of 0.2% and incubate on ice for 3 minutes to allow permeation.

[0065] 4. Set the horizontal centrifuge to 4°C. All centrifugation steps below shall be performed at 4°C. After the reaction is complete, centrifuge at 500g for 5 minutes and collect the cell nuclear pellet.

[0066] 5. Remove the supernatant and redissolve the nucleus pellet with 800 μL 1×PBS (1% BSA) to further wash away the previous reaction solution. Centrifuge at 500g for 5 minutes to collect the nuclei.

[0067] 6. Mix 100 μM hybridization probes in equal proportions, heat at 90 °C for 5 minutes in a PCR instrument, and then quickly place on ice to quench.

[0068] 7. Prepare the probe hybridization reaction solution according to the reaction conditions shown in Table 1, resuspend the cell nuclei in it, and incubate at 40°C for 1 hour.

[0069] Table 1: Probe Hybridization Reaction Solution

[0070] Components Dosage (μL) 20×SSC buffer 10 100% Tween 20 2 Fish sperm DNA (5mg / ml) 4 200mM RVC 20 RNAsin Plus (40 U / μL) 0.2 Hybridization probe (100μM) 8 / Genes

[0071] 8. After the hybridization reaction, in order to wash away the unbound probe, add 500 μL of washing buffer (1X PBS (1% BSA)) to the sample, mix well, and centrifuge at 500g for 5 minutes to collect the cell nuclei.

[0072] 9. Repeat step 8 twice, then prepare the post-hybridization washing buffer according to Table 2, and resuspend the cell nuclei in the post-hybridization washing buffer and incubate at 40°C for 20 minutes.

[0073] Table 2: Washing buffer after hybridization

[0074]

[0075]

[0076] 10. After incubation, repeat step 8 twice. At the same time, prepare the backbone and insert sequence hybridization buffer according to Table 3, and resuspend the cell nuclei in the backbone and insert sequence hybridization buffer and incubate at 37°C for 30 minutes.

[0077] Table 3: Hybridization buffers for backbone and insert sequences

[0078] Components Dosage (μL) 20×SSC buffer 10 Backbone DNA (10 μM) 0.4 Inserted DNA (10 μM) 0.4 RNAsin Plus (40 U / μL) 0.2 Ultrapure water 189 Total reaction volume 200

[0079] 11. Set the incubator temperature to 21℃ in advance. After incubation, repeat step 8 three times. At the same time, prepare the ligation buffer according to Table 4, resuspend the cell nuclei in the ligation buffer, and blow them evenly to prevent cell nuclei from clumping. Incubate at 21℃ for 30 minutes.

[0080] Table 4: Ligation Buffer

[0081] Components Dosage (μL) 10×T4 ligation buffer 20 T4 DNA ligase (5 U / μL) 0.2 RNAsin Plus (40 U / μL) 0.2 Ultrapure water 179.6 Total reaction volume 200

[0082] 12. After incubation, repeat step 8 twice. At the same time, prepare the rolling circle replication reaction buffer according to Table 5, and resuspend the cell nuclei in the rolling circle replication reaction buffer and incubate at 30°C for 2 hours.

[0083] Table 5: Rolling Loop Replication Reaction Buffer

[0084] Components Dosage (μL) 10×Phi29 DNA polymerase buffer 20 Phi29 DNA polymerase (10 U / μL) 2 dNTPs (10mM) 4 RNAsin Plus (40 U / μL) 0.2 Ultrapure water 173.8 Total reaction volume 200

[0085] 13. After incubation, repeat step 8 twice. At the same time, prepare the fluorescent probe hybridization buffer according to Table 6, resuspend the cell nuclei in the fluorescent probe hybridization buffer, and incubate at 37°C for 30 minutes.

[0086] Table 6: Fluorescent probe hybridization buffer

[0087] Components Dosage (μL) 1×PBST 998 DNA fluorescent probe (100μM) 1 RNAsin Plus (40 U / μL) 1 Total reaction volume 1000

[0088] 14. After incubation, repeat step 8 twice, resuspend the cell nuclei in 300 μL 1×PBS (5 μg / mL DAPI), and sort the fluorescently positive cell nuclei on a flow cytometer.

[0089] 15. Filter the cell suspension using a 70 μm cell filter and transfer the cell suspension to a flow cytometry tube.

[0090] 16. Place the sample loading tube into the sample loading slot of the flow cytometer, adjust the voltage of different fluorescence channels, remove cell debris according to cell complexity (SSC) and cell size (FSC), and classify single cells or single-nucleus populations according to the fluorescence intensity of the nuclear dye DAPI.

[0091] 17. Based on the intensity of the fluorescent probe of the marker gene, the target cell population is divided and sorted into 96-well plates containing collection solution for subsequent library construction steps.

[0092] III. ATAC-seq Database Creation Process

[0093] 1. Prepare the Tn5 transposase reaction mixture according to Table 7. Resuspend the cells or nuclear pellet before RNA FISH fixation, and react at 37°C for 30 minutes in a PCR instrument. After fragmenting the genome, fix the cells and continue the RNA FISH operation as described above until the cells are sorted by flow cytometry.

[0094] Table 7: Tn5 transposase reaction mixture system

[0095] Components Dosage (μL) 5×TTBL 1.0 TTE MIX TD501 0.5 100% DMF 0.5 0.5% Digitonin 0.1 2.5% IGEPAL-630 0.2 2.5% Tween 20 0.2 1X PBS 1.65 Ultrapure water 0.85 Total reaction volume 5.0

[0096] 3. Use 10 μL PBS solution as the collection medium to collect the target cell nuclei separated by flow cytometry. Generally, collect 500 target cell nuclei for each cell population.

[0097] 4. Prepare a 2×Tn5 transposase reaction stop solution with the components shown in Table 8. Add an equal volume of the reaction stop solution to the sample, mix well and avoid generating bubbles, and then place the sample in a PCR instrument and react at 55℃ for 12 hours.

[0098] Table 8: Termination solution for 2×Tn5 transposase reaction

[0099]

[0100]

[0101] 4. Remove the DNA magnetic beads stored at 4℃, vortex to mix, and equilibrate at room temperature for 30 minutes.

[0102] 5. After the reaction is terminated, add 36 μL of DNA magnetic beads to the sample, mix well by pipetting, and let stand at room temperature for 10 minutes.

[0103] 6. Place the sample on the magnetic rack and let it stand at room temperature for 5 minutes until the magnetic beads are completely adsorbed onto the tube wall. Then, gently aspirate the supernatant with a pipette.

[0104] 7. Add 100μL of 80% ethanol to clean the magnetic beads, remove the ethanol, repeat the cleaning steps, remove the ethanol solution, and let it air dry at room temperature for 5 minutes.

[0105] 8. Prepare the ATAC-seq library PCR amplification reaction solution. The composition of the reaction solution is shown in Table 9. Then, resuspend the magnetic beads in the reaction solution, mix well, and place them in the PCR instrument to run the ATAC-seq PCR amplification program in Table 10.

[0106] Table 9: ATAC-seq library PCR amplification reaction solution

[0107] Components Dosage (μL) 2×KAPA HiFi premixed liquid 12.5 F(25μM) 1.25 R(25μM) 1.25 Total reaction volume 15

[0108] Note: The F and R primers contain tag sequences that identify the sample. The F and R primers added to each sample well have different tag sequences to distinguish them.

[0109] Table 10: ATAC-seq PCR amplification program

[0110] temperature time Cycle number 72℃ 5min 1 98℃ 3min 1 98℃ 20s 17 63℃ 30s 17 72℃ 3min 17 4℃ ∞

[0111] Note: The number of amplification cycles varies depending on the number of cells in the reaction; typically, 500 cells require 17 amplification cycles.

[0112] 10. After the PCR reaction is complete, add 45 μL of DNA magnetic beads to the product and let it stand at room temperature for 10 minutes.

[0113] 11. Place the sample on the magnetic rack and wait until the magnetic beads are completely adsorbed onto the side wall of the centrifuge tube. Gently aspirate the supernatant with a pipette. Add 100 μL of 80% ethanol to wash the magnetic beads and remove the ethanol. Repeat once and then air dry at room temperature.

[0114] 12. Resuspend the magnetic beads in 20 μL of ultrapure water, blow them evenly, and let them stand at room temperature for 10 minutes.

[0115] 13. Place the sample on the magnetic rack and wait until the magnetic beads are completely adsorbed onto the side wall of the centrifuge tube. Then, gently aspirate the supernatant with a pipette and transfer it to a new PCR tube.

[0116] 14. Use the Qubit 3.0 instrument to determine the sample concentration, dilute all samples to 5 ng / μL, take an equal volume of the diluted samples, mix them thoroughly, and perform next-generation sequencing.

[0117] IV. ACC-seq Database Construction Process

[0118] Fresh group operation steps:

[0119] The library preparation and RNA FISH sorting process is the same as the ATAC-seq process described above.

[0120] Formaldehyde fixation group operation steps

[0121] 1. Resuspend the cell nuclei in 40 μL of 1×PBS, then immediately add an equal volume (40 μL) of pre-chilled 2% paraformaldehyde (PFA, w / v), mix thoroughly (pipe 5–10 times), and fix on ice for 10 minutes.

[0122] 2. Add 16 μL of 1M Tris-HCl (pH 7.5, final concentration 166 mM) to the sample and incubate at room temperature for 10 minutes to terminate the crosslinking reaction.

[0123] 3. Add 150 μL of pre-cooled PBSTR (containing 1% BSA and 4 U / mL RNAsin Plus) buffer, mix thoroughly (pipe and vortex 5-10 times), centrifuge at 500g, 4℃ for 3 minutes, and discard the supernatant.

[0124] 4. Resuspend the cell nuclei in 250 μL of pre-cooled PBSTR, centrifuge at 500g and 4℃ for 3 minutes, and discard the supernatant.

[0125] 5. Resuspend the cell nuclei in 40 μL of Tn5 transposase reaction mixture (same as the fresh group) and incubate at 37°C for 1 hour to perform Tn5 cleavage reaction.

[0126] 6. After the Tn5 reaction is complete, add 200 μL of pre-cooled PBSTR buffer, mix thoroughly (pipe 5–10 times), filter through a 40 μm filter and transfer the cell nuclei to a 1.5 mL centrifuge tube.

[0127] 7. Centrifuge at 500g, 4℃ for 3 minutes, and discard the supernatant.

[0128] 8. Subsequent experimental procedures are the same as those after RNA FISH fixation.

[0129] Procedure for the 1,6-hexanediol (Hex) treatment group:

[0130] 1. Resuspend the cell nuclei in 40 μL of 10% 1,6-hexanediol solution (prepared in 1×PBS) and incubate at room temperature for 2 minutes.

[0131] 2. Immediately add an equal volume (40 μL) of pre-cooled 2% paraformaldehyde (PFA, w / v), mix thoroughly (blow 5-10 times), and fix on ice for 10 minutes.

[0132] 3. Add 16 μL of 1M Tris-HCl (pH 7.5, Invitrogen) to the sample. TM 15567027 (final concentration 166mM), incubated at room temperature for 10 minutes to terminate the crosslinking reaction.

[0133] 4. Add 150 μL of pre-cooled PBSTR buffer, mix thoroughly (pipe and vortex 5-10 times), centrifuge at 500g, 4℃ for 3 minutes, and discard the supernatant.

[0134] 5. Resuspend the cell nuclei in 250 μL of pre-cooled PBSTR (containing 1% BSA and 4 U / mL RNAsin Plus), centrifuge at 500g and 4℃ for 3 minutes, and discard the supernatant.

[0135] 6. Resuspend the cell nuclei in 40 μL of Tn5 reaction buffer (same as the fresh group) and incubate at 37°C for 1 hour to perform the Tn5 tagging reaction.

[0136] 7. After the Tn5 reaction is complete, add 200 μL of pre-cooled PBSTR buffer, mix thoroughly (pipe 5-10 times), filter through a 40 μm filter, and transfer the cell nuclei to a 1.5 mL centrifuge tube.

[0137] 8. Centrifuge at 500g, 4℃ for 3 minutes, and discard the supernatant.

[0138] 9. Subsequent experimental procedures are the same as those after RNA FISH fixation.

[0139] V. ACC-seq Data Analysis

[0140] 1. Preprocessing of sequencing data

[0141] The raw sequencing data reads were input into the cutadapt software (version 1.18), and the parameters were set as follows: minimum retention length -m 20, maximum error rate -e 0.1, minimum adapter match length -O 3, low quality base trimming threshold -q 20, and quality score base set to 33 (--quality-base=33) to remove sequencing adapter contamination sequences and low quality bases.

[0142] 2. Genome alignment

[0143] Bowtie2 alignment software (version 2.3.3.1) was used to align the preprocessed sequencing reads with reference genomes (e.g., mouse genome mm10 or human genome hg38). The alignment parameters set included: maximum allowable mismatches - N 1, seed length - L 25, and mixed alignment and non-discordant alignment were disabled (--no-mixed, --no-discordant) to ensure alignment accuracy.

[0144] 3. Removal of duplicate segments

[0145] The MarkDuplicates module in Picard (version 2.20.4) was used to label repetitive reads generated during PCR amplification. Subsequently, Samtools software (version 1.6) with the filter parameter set to -F 1024 was used to remove the labeled repetitive sequences, thereby improving the accuracy of the analyzed data.

[0146] 4. Signal file generation

[0147] Use the deepTools tool to convert the deduplicated alignment results into Bigwig format for subsequent visualization and signal strength comparison analysis.

[0148] 5. Peak Region Identification

[0149] MACS2 (version 2.1.2) was used to identify significant signal-enriched regions across the genome, including annotations of functional regions such as open chromatin regions and transcription factor binding regions.

[0150] 6. Calculation of peak differences

[0151] The number and location of differential peaks between the Fresh group, the formaldehyde fixation group, and the hexanediol (Hex) treatment group were calculated and obtained using diffbind.

[0152] Results and Discussion

[0153] The results of this invention are shown in Figures 2-5 In, among them, Figure 2 The figure shows RNA FISH staining and flow cytometry sorting of cell lines and mouse hippocampus according to embodiments of this application. In this figure, a. Results of RNA FISH staining for the Tyr gene probe in mouse cell lines; the left panel shows B16 cells with high Tyr mRNA expression, and the right panel shows 4T1 cells with low Tyr mRNA expression. b. Flow cytometry analysis results of human CLU gene staining in HeLa (human) and NIH / 3T3 (mouse) cell nuclei; scale bar is 10 μm. c. Mouse hippocampal staining results; P1 represents Tubb3-positive nuclei, P2 represents Tubb3-positive and Arc-positive nuclei, and P3 represents Tubb3-positive and Arc-negative nuclei. d. qPCR quantification of gene expression levels in nuclei sorted from different phyla in figure c. Flow cytometry sorted 100 nuclei for reverse transcription and qPCR quantification; the Y-axis represents the absolute Cq value of gene expression. Figure 3The quality of RNA FISH-enriched nuclear ATAC-seq data according to embodiments of this application is shown. In this figure, a. IGV screenshots of signal peaks after alignment of single-nucleus ATAC-seq data with the genome. b. Statistical graph of the fraction of sequencing reads in signal peaks (FRiP) in bulk (100 nuclei) and single-nucleus ATAC-seq data. c. Changes in chromatin accessibility at the Arc gene location in RNA FISH-enriched mouse hippocampal Arc-positive neurons. Figure 4 A schematic diagram of the ACC-seq technology according to an embodiment of this application is shown. In this diagram, the method utilizes paraformaldehyde as a cross-linking agent to detect chromatin-associated aggregate structures. In the native state of cells, the Tn5 transposase can enter chromatin, including regions occupied by aggregates. However, after paraformaldehyde fixation, the cross-linked aggregates restrict Tn5's cleavage and insertion of DNA, thereby inhibiting transcriptase reactions in that region. If cells are treated with 1,6-hexanediol (1,6-Hex) before fixation, these liquid-liquid phase separation aggregates can be dissolved, releasing the chromatin structures within, thereby enhancing Tn5's entry and tagging response in the relevant regions. Through subsequent high-throughput sequencing and chromatin accessibility analysis, different regions in the genome can be divided into two categories: hexanediol-sensitive regions: sensitive to 1,6-Hex treatment, indicating that their chromatin accessibility is significantly affected by aggregates; and hexanediol-insensitive regions: insensitive to 1,6-Hex treatment, with minimal impact from aggregates. Figure 5 The image shows a screenshot of the IGV signal at the Junb gene location in mouse hippocampal neurons according to an embodiment of this application via ACC-seq. In this figure, the cell population consists of mouse hippocampal neurons labeled with the Tubb3 probe, and ACC-seq library construction and analysis were performed. The red box marks the chromatin region near the Junb gene that is sensitive to 1,6-hexanediol.

[0154] The inventor's technical principles are as follows Figure 1 As shown in Figure a, single cells or single cell nuclei isolated from tissue are first subjected to Tn5 transposase to break down the genome in vitro, followed immediately by formaldehyde fixation of the nuclei to prevent DNA fragment leakage. Then, RNA FISH technology is used to label the target cell population based on nucleus-specific RNA molecules. The target cells are then obtained through flow cytometry sorting and decrosslinking treatment, followed by ATAC-seq library construction and amplification. The principle of RNA FISH technology is as follows: Figure 1As shown in b, this technology is based on PLAYR (Proximity Ligation Assay for RNA). It involves designing multiple pairs of adjacent probe molecules that bind to the target RNA. The probe molecules contain fixed sequences at their ends, providing binding sites for the backbone DNA molecule and the insert DNA molecule. When the backbone DNA molecule and the insert DNA molecule bind to the probe, T4 DNA ligase ligates them into a single-stranded circular DNA molecule. This circular DNA molecule is then extended into a long single-stranded DNA molecule by phi29 DNA polymerase. The sequence of this long single-stranded DNA molecule is a continuous repetition of the circular DNA molecule sequence. Therefore, by designing a fluorescent probe with the same sequence as the insert DNA molecule, the fluorescent probe can be enriched on the target RNA molecule.

[0155] First, the inventors verified the specificity of RNA FISH staining in cell lines, such as... Figure 2 As shown in Figure a, the inventors designed probes targeting the Tyr gene to stain two different cell types, B16 and 4T1. B16 cells highly express the Tyr gene, while 4T1 cells do not. Therefore, the inventors observed a significant fluorescence signal in B16 cells, while 4T1 cells showed almost no fluorescence signal, indicating that this RNA FISH technique has high staining efficiency and a high signal-to-noise ratio. Similar experiments were conducted on cell nuclei, using the CLU gene probe to stain HeLa cells and NIH / 3T3 cells, respectively. Flow cytometry analysis of the staining results revealed that HeLa cells highly express the CLU gene. Therefore, in the flow cytometry analysis, the fluorescence intensity of HeLa cells was significantly stronger than that of NIH / 3T3 cells. This result also demonstrates that the inventors' RNA FISH labeling and sorting technology can screen for target cell populations using flow cytometry.

[0156] The inventors further tested this technology in highly heterogeneous tissues. Adult mice were stimulated with 18mA electroconvulsive therapy to activate hippocampal neurons. One hour later, the hippocampal tissue was dissected and used for RNA FISH and ATAC-seq library construction. Activated hippocampal neurons rapidly express several Immediate Early Genes (IEGs) to activate downstream synaptic activity and other biological processes. Arc is a typical IEG, and it is significantly highly expressed after hippocampal neuron activation. The inventors used RNA FISH to label the Tubb3 and Arc genes. Tubb3 positivity indicates neurons, while cells positive for both Tubb3 and Arc indicate activated neurons. Flow cytometry clearly distinguishes Arc-positive and Arc-negative cells. Figure 2As shown in Figure c, when the inventors performed qPCR quantification on the different cell groups obtained from the analysis, they found that the Cq value of Arc mRNA in Arc-positive cells was much lower than that in Arc-negative cells, even differing by more than 10 cycles, while the expression level of Gapdh mRNA did not differ significantly between the two cell groups. This result again demonstrates that this technology can be efficiently applied to the sorting of target cell groups in complex tissues.

[0157] Before performing RNA FISH, the inventors needed to fix cells or cell nuclei with paraformaldehyde (PFA) before staining. Paraformaldehyde is a widely used cross-linking agent for cell fixation, which can preserve the interactions between intracellular molecules, thereby preventing cell death, damage, and degradation. However, for capturing chromatin openness, the inventors typically used ATAC-seq, a technique that uses the Tn5 transposase to break down DNA in open regions of the genome. However, paraformaldehyde-fixed cells or nuclei affect the efficiency of Tn5 transposase penetration and access to chromatin, and the cross-linking of DNA molecules with DNA-binding proteins by formaldehyde molecules also affects the efficiency of transposase in breaking down the genome, significantly reducing the data quality of ATAC-seq.

[0158] To address the impact of PFA fixation on ATAC-seq, the inventors employed a technique involving Tn5 transposase to fragment the chromatin in all cell nuclei before paraformaldehyde fixation. Immediately after this treatment, paraformaldehyde was used to fix the nuclei, immobilizing the fragmented DNA within the nuclei. Subsequently, RNA FISH was used to label the target cell populations. The sorted nuclei were then decross-linked, releasing the fragmented DNA, which will be used for subsequent library construction in ATAC-seq. Figure 1 As shown in figure a, single-nucleus ATAC-seq also exhibits a high signal-to-noise ratio and reproducibility, with clear signal peaks observed in regions such as gene promoters. Although the inventors can obtain high-quality single-nucleus ATAC-seq data, a large number of nuclei (100 nuclei) can provide more effective information, and the proportion of sequencing reads falling on ATAC-seq signal peaks (FRiP) is significantly higher than that of single-nucleus data. Figure 3 As shown in b. Therefore, a large amount of ATAC-seq data from cell nuclei will more effectively help the inventors study changes in chromatin openness in specific biological processes. Furthermore, for the activated Arc-positive neurons mentioned above, the chromatin openness in the promoter region of the Arc gene is significantly increased compared to unactivated neurons, reflecting the high expression of the Arc gene in Arc-positive neurons, further demonstrating the accuracy of the inventors' enrichment sequencing technology, such as... Figure 3 As shown in c.

[0159] The inventors then tested the ACC-seq technology based on RNA FISH sorting, and the detailed technical principles are as follows: Figure 4 The dissociated single-cell or single-nuclear suspensions were divided into three groups: the first group (Fresh group) involved cells first undergoing Tn5 transposase fragmentation followed by formaldehyde fixation to obtain chromatin openness information in the original cellular state; the second group (Formaldehyde-fixed group) involved cells first undergoing formaldehyde fixation to immobilize condensates on chromatin, followed by Tn5 transposase fragmentation; and the third group (1,6-hexanediol-treated group) involved cells first undergoing 1,6-hexanediol treatment to disrupt condensates on the chromatin, releasing the chromatin regions previously enclosed by condensates, followed by formaldehyde fixation and then Tn5 transposase fragmentation of the chromatin. All three groups of cells were then subjected to parallel RNA FISH staining to label and sort the target cell populations, thereby achieving ACC-seq of the target cell types at the tissue level. After sequencing, the differences in chromatin openness signals among the three groups of cells were calculated using bioinformatics to identify 1,6-hexanediol-sensitive condensate-covered regions and 1,6-hexanediol-insensitive regions on the chromatin.

[0160] The inventors used RNA FISH to sort hippocampal neurons from resting mice using the Tubb3 gene probe for ACC-seq. RNA FISH helped the inventors obtain target neurons from highly heterogeneous tissues, improving cell purity. The inventors obtained high-quality ACC-seq data using only about 3000 cells. Figure 5 As shown, a 1,6-hexanediol-sensitive signal peak is observed at the Junb gene location. Junb is an immediate early gene that can respond to neuronal stimulation and mediate neuronal function.

[0161] In this invention, unless otherwise expressly specified and limited, the terms "first" and "second" are used for descriptive purposes only and should not be construed as indicating or implying relative importance or implicitly specifying the number of indicated technical features. Thus, a feature defined as "first" or "second" may explicitly or implicitly include at least one of that feature. In the description of this invention, "a plurality of" means at least two, such as two, three, etc., unless otherwise expressly and specifically limited.

[0162] In the description of this specification, the references to terms such as "one embodiment," "some embodiments," "example," "specific example," or "some examples," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples. Moreover, without contradiction, those skilled in the art can combine and integrate the different embodiments or examples described in this specification, as well as the features of different embodiments or examples.

[0163] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention.

Claims

1. A method for constructing a sequencing library, characterized in that, The sequencing library is used for cell-specific ATAC-seq chromatin open sequencing, and the method includes: Obtain a suspension containing cell units, said cell units including at least one of a single cell nucleus and a single cell, said cell units containing a target nucleic acid sequence; The genome of the cell unit was fragmented using Tn5 transposase; The cell units that have undergone the aforementioned disruption treatment are immobilized using a polyoxymethylene solution; The cell units that have undergone the immobilization treatment are then subjected to fluorescent labeling. Based on the fluorescence, the cell samples are sorted to obtain the desired cell units; The desired cell unit is subjected to decrosslinking treatment; Cellular units that have undergone decrosslinking treatment are subjected to PCR amplification using primers carrying sequencing adapters to obtain the sequencing library. The fluorescent labeling process includes: The first probe and the second probe are specifically bound to the target nucleic acid sequence, and the binding site of the first probe and the binding site of the second probe are spaced apart by a predetermined length. An insert, a backbone, and a ligase are provided, wherein the nucleic acid sequences of the insert and the backbone are configured such that when the first and second probes specifically bind to the target nucleic acid sequence, the insert and the backbone can be ligated into a single-stranded loop. The single-chain ring is subjected to rolling ring replication to obtain a long single-chain molecule, the long single-chain molecule containing multiple repeating units; and The third probe is combined with the repeating unit, and the third probe carries a detectable signal.

2. The method according to claim 1, characterized in that, The decrosslinking treatment is performed by high-temperature treatment using a solution containing proteinase K and sodium dodecyl sulfate.

3. The method according to claim 1, characterized in that, The target nucleic acid sequence is RNA; optionally, the target nucleic acid sequence is mRNA. Optionally, the first probe, the second probe, the third probe, the insert fragment, and the backbone fragment are each independently DNA; Optionally, the detectable signal is a fluorescence signal; Optionally, the sorting is performed using a flow cytometer.

4. The method according to claim 1, characterized in that, The cell unit contains multiple different target nucleic acid sequences, each of which is independently assigned the same or different detectable signals; Optionally, the cell unit contains at least five different target nucleic acid sequences, such as eight different target nucleic acid sequences; Optionally, the multiple different target nucleic acid sequences originate from the same gene; Optionally, there are no overlapping regions between the multiple different target nucleic acid sequences.

5. The method according to claim 1, characterized in that, The binding site of the first probe and the binding site of the second probe are separated by no more than 10 bp, for example, the separation is no more than 5 bp, and preferably the separation is 3 to 5 bp; Optionally, the lengths of the first probe and the second probe are each independently 15 to 25 bp, preferably about 20 bp; Optionally, the Tm values ​​of the first probe and the second probe are each independently 58–60 degrees Celsius; Optionally, the GC content of the first probe and the second probe are each independently 30% to 70%.

6. The method according to claim 1, characterized in that, The cell units are immobilized before the first and second probes specifically bind to the target nucleic acid sequence. Optionally, the immobilization treatment is performed using polyoxymethylene; Optionally, the immobilization treatment is performed using a 37% polyoxymethylene solution.

7. The method according to claim 1, characterized in that, The ligase is T4 DNA ligase, and the rolling circle replication is performed using Phi29 DNA polymerase.

8. A method for constructing a sequencing library, characterized in that, The sequencing library is used for cell-specific ACC-seq chromatin open sequencing, and the method includes: Obtain a suspension containing cell units, said cell units including at least one of a single cell nucleus and a single cell, said cell units containing a target nucleic acid sequence; The suspension was divided into a Fresh group, a formaldehyde fixation group, and a 1,6-hexanediol treatment group. The Fresh group, formaldehyde-fixed group, and 1,6-hexanediol-treated group were subjected to their respective chromatin-breaking treatments, using Tn5 transposase to break down the chromatin. The cell units that have undergone the aforementioned disruption treatment are immobilized using a polyoxymethylene solution; The cell units that have undergone the immobilization treatment are then subjected to fluorescent labeling. Based on the fluorescence, the cell samples are sorted to obtain the desired cell units; The desired cell unit is subjected to decrosslinking treatment; Cellular units that have undergone decrosslinking treatment are subjected to PCR amplification using primers carrying sequencing adapters to obtain the sequencing libraries, which include Fresh sequencing libraries, formaldehyde-fixed sequencing libraries, and 1,6-hexanediol-treated sequencing libraries. The fluorescent labeling treatment is as described in claim 1. in, The disruption treatment of the Fresh group was carried out by reacting the cell units of the Fresh group with a reaction mixture containing Tn5 transposase. The interruption process of the formaldehyde fixation group includes: The cell units of the formaldehyde-fixed group were fixed with formaldehyde using a polyoxymethylene solution; and The formaldehyde-fixed cell units were reacted with a reaction mixture containing Tn5 transposase to break down the chromatin of the cell units. The interruption process of the 1,6-hexanediol treatment group includes: The cell units in the 1,6-hexanediol treatment group were treated to disrupt chromatin aggregates and release chromatin regions encased in aggregates. The treated cell units were then subjected to formaldehyde fixation using a polyoxymethylene solution; and The formaldehyde-fixed cell units were reacted with a reaction mixture containing Tn5 transposase to break down the chromatin of the cell units.

9. A method based on enriched cell-specific chromatin open sequencing technology, characterized in that, include: For the sample to be tested, a sequencing library is constructed according to the method described in any one of claims 1 to 8; The sequencing library was sequenced; The sequencing results are analyzed to obtain information on the chromatin accessibility of the sample to be tested.

10. The method according to claim 9, characterized in that, The sample to be tested is an activated neuron from the hippocampus. The target nucleic acid sequence used in the sorting process comes from at least one of the genes Tubb3 and Arc. The sorting uses Tubb3 positivity and Arc positivity as the sorting criteria for the activated neurons.

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