Method for inhibiting nuclease activity, method for separating cell nucleus from cell, and method for extending DNA

Abstract

The present disclosure relates to a method of inhibiting nuclease activity in a biological sample. In accordance with certain embodiments of the present disclosure, the method comprises mixing a biological sample with a metal-chelating agent complex. The present disclosure also discloses a method of isolating a cell nucleus from a cell in a biological sample by using the metal-chelating agent complex, and a method of adding a deoxyribonucleotide to the 3'terminal of a deoxyribonucleic acid.

Description

Technical field

[0001] The present disclosure relates to the field of molecular biology. Specifically, the present disclosure relates to methods for inhibiting nuclease activity, methods for isolating cell nuclei from biological samples, and methods for extending DNA. [Background Technology]

[0002] Nucleic acid amplification (NAA) is crucial for achieving high detection sensitivity in clinical and biological samples. Currently, there are two main strategies for NAA: in vitro transcription and polymerase chain reaction (PCR). The in vitro transcription process requires fragile complementary deoxyribonucleic acid (cDNA) as an intermediary and suffers from low double-stranded DNA (dsDNA) synthesis efficiency. Furthermore, in vitro transcription involves complex purification steps, which inevitably lead to sample loss. In contrast, PCR amplification is a more common and straightforward technique, requiring fewer steps and not requiring RNA generation. However, PCR requires the addition of a handle to the 3' end of the DNA. Handles are generated by enzymatic fragmentation followed by dsDNA synthesis, polymerization using random primers carrying the handle, template exchange, or the use of homologous polymerases. Enzymatic fragmentation followed by dsDNA synthesis not only suffers from low dsDNA synthesis efficiency but also suffers from sequence bias during the enzymatic fragmentation process. Polymerization reactions using random primers with handles also have problems with sequence bias due to random primer binding preferences, secondary structure of the target DNA, and the need to remove primers to avoid interference with subsequent PCR amplification. Template exchange or the use of homologous polymerases to add PCR handles in one step, followed by amplification without purification, can maximize the detectable DNA fragments. However, template exchange may be due to the lack of deoxygenase activity. Cytidine nucleotides are incorporated into DNA tails, resulting in poor efficiency. Homopolymerization reactions using terminal deoxynucleotidyl transferase (TdT) are not ideal in current reaction systems. Specifically, because recessed DNA ends are located on complementary mRNA or within secondary structures, these methods currently fail to fully extend the recessed DNA ends. Therefore, there is an urgent need in the art for a reaction system that can fully tailor all DNA substrates, enabling DNA amplification without purification.

[0003] Nucleases are ubiquitous in the environment and biological samples, causing nucleic acid degradation and hindering nucleic acid testing in clinical trials and research. Ribonucleases (RNases) are among the most challenging nucleases to detect due to their ubiquitous presence in the environment and biological samples, their metal ion dependence, and their heat tolerance. Current methods for inhibiting RNase activity have significant limitations, particularly when manipulating delicate samples such as nuclei in tissues. For example, low pH denatures proteins, alters nuclear structure, and degrades nucleotides. Vanadyl ribonucleosides contain divalent cations that interfere with downstream enzyme activity. While they exhibit similar functionality to recombinant RNase inhibitors, these nucleotide analogs are not readily available and lack inhibitory efficacy against samples containing different types of nucleases, particularly DNases. Reducing agents, on the other hand, require prolonged or high-temperature treatment, precluding one-pot lysis, which would immediately expose the RNA to nucleases. Highly negatively charged polymers (e.g., polyethylene sulfonic acid) are used to bind to RNase and inactivate it. Highly negatively charged polymers will also competitively bind to positively charged nuclear proteins, causing the unfixed nuclear structure to deform. In addition, these large polymers are not easy to remove from the extracted nuclei, complicating downstream enzyme reactions. Finally, diethyl pyrocarbonate (DEPC) carboxymethylates RNA purines and reacts with amino acid side chains (especially lysine), reacting with formaldehyde and N-hydroxysuccinimide esters. ester, NHS ester) and other cross-linking agents compete for effective cross-linking. 2+ It has been shown to inhibit RNase activity, but is not applicable in most biological systems because (1) Cu 2+ In a neutral pH environment, an insoluble precipitate (Cu(OH)2) is formed; (2) Cu 2+ By interacting with multiple histidine residues and cross-linking with proteins, it causes protein coagulation. 2+ It cannot be used alone as an RNase or nuclease inhibitor because Cu 2+ The concentration required to effectively inhibit the hydrolytic activity is much higher than that of soluble Cu 2+As described above, there is a pressing need in the art for a non-aggregating and soluble reagent that can be used to protect nucleic acids (including RNA and DNA) in biological samples without significantly altering the native biological environment (e.g., pH, ionic conditions, and temperature) or causing unnecessary covalent modifications.

[0004] Profiling nucleic acid expression in single cells or nuclei is an increasingly important goal. Upon cell lysis in multicellular organisms, RNA and DNA are immediately exposed to nucleases in cellular vesicles and extracellular fluids (e.g., plasma). While single cells can be dissociated from tissue for intact cell analysis, tissue-dependent processing inevitably introduces artifacts that hinder cross-tissue comparisons of cells of the same type. Furthermore, dissociation can result in biased release among different cell types, and ex vivo culture is associated with altered cell states. Therefore, direct isolation of nuclei from pulverized tissue can circumvent these issues by providing appropriate sensitivity and using nuclei as input to classify cell types. These methods substantially expand the acceptable sample range. However, in certain tissues rich in nucleases, such as the blood-rich spleen or exocrine pancreas, nuclear nucleic acids are rapidly degraded in the lysate. This difficulty is demonstrated by the lack of a database for one-pot lysis of the spleen and the specific low-pH buffer requirements of pancreatic acinar cells.

[0005] In view of this, the field still needs to continue to develop methods for protecting nucleic acids in biological samples (especially RNA in the cell nucleus) and effectively amplifying original or converted DNA (especially those with inaccessible recessed ends) to achieve the purpose of sensitive detection. [Summary of the invention]

[0006] The present invention summary is intended to provide a simplified summary of the present invention so that readers can have a basic understanding of the present invention. This invention summary is not a complete overview of the present invention and is not intended to identify important / critical components of the present invention or to define the scope of the present invention.

[0007] A first aspect of the present disclosure relates to a method for inhibiting nuclease activity in a biological sample, wherein the nuclease comprises deoxyribonuclease (DNase) and ribonuclease (RNase). According to certain embodiments of the present disclosure, the method comprises mixing the biological sample with a metal ion-chelator complex to inhibit the nuclease activity in the biological sample.

[0008] According to certain embodiments of the present disclosure, the metal ion-chelator complex is copper citrate (Cu-Citrate), copper nitrilotriacetic acid (Cu-NTA) or copper iminodiacetic acid (Cu-IDA).

[0009] According to a preferred embodiment of the present disclosure, the molar ratio of the metal ion to the chelating agent in the metal ion-chelating agent complex is greater than or equal to 1. More preferably, the molar ratio of the metal ion to the chelating agent in the metal ion-chelating agent complex is equal to 1.

[0010] A second aspect of the present disclosure relates to a method for isolating cell nuclei from cells in a biological sample using a metal ion-chelator complex. According to certain embodiments of the present disclosure, the method comprises the following steps: (a) mechanically disrupting cells in the biological sample; (b) In the presence of metal ion-chelator complexes (e.g., Cu-Citrate, Cu-NTA, or Cu-IDA) mixing the product of step (a) with a lysis buffer to release the cell nucleus from the cell; and (c) isolating cell nuclei from the product of step (b), thereby producing isolated cell nuclei.

[0011] According to certain exemplary embodiments of the present disclosure, in step (c) of the method, the cell nucleus is isolated by the following steps: (c-1) treating the product of step (b) by concentration gradient centrifugation in the presence of a metal ion-chelating agent complex; and (c-2) Collecting a fraction containing the cell nucleus from the product of step (c-1).

[0012] According to various embodiments of the present disclosure, the lysis buffer of step (b) comprises 0.1-3% (v / v) of a nonionic surfactant. In one embodiment of the present disclosure, the lysis buffer of step (b) comprises 0.1% (v / v) of polyoxyethylene sorbitan monooleate (polysorbate 20) and 0.1% (v / v) of nonylphenol polyoxyethylene ether (NP-40).

[0013] According to certain embodiments of the present disclosure, the metal ion-chelator complex is Cu-Citrate, wherein the concentration of Cu-Citrate in the lysis buffer is between about 2-100 mM. In one embodiment of the present disclosure, the concentration of Cu-Citrate in the lysis buffer is 10 mM.

[0014] According to certain embodiments of the present disclosure, the method further comprises: (d) a mixture containing methanol and magnesium ions (Mg 2+ ) solution to fix the cell nucleus of step (c). In some preferred embodiments of the present disclosure, the solution contains Mg 2+ The concentration is 5 mM.

[0015] A third aspect of the present disclosure relates to a method for adding a deoxyribonucleotide to the 3' end of DNA. The method comprises: (a) mixing DNA with a reaction buffer containing TdT, deoxyribonucleotides and transition metal ions, The reaction buffer does not contain metal ions other than transition metal ions (for example, potassium ions (K + ), magnesium ions (Mg 2+ ), sodium ion (Na + ) etc.); and (b) Incubating the mixture of step (a) at 37° C. for 30-120 minutes to produce DNA having a deoxyribonucleotide at the 3′ end.

[0016] According to certain embodiments of the present disclosure, the transition metal ion is a cobalt ion (Co 2+ ) or manganese ions (Mn 2 + ).

[0017] According to certain embodiments of the present disclosure, the deoxyribonucleotide is deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyuridine triphosphate (dUTP). According to certain exemplary embodiments, the deoxyribonucleotide is dATP, and the concentration of dATP in the reaction buffer is between 0.5 and 10 mM. In one specific embodiment, the concentration of dATP in the reaction buffer is 2 mM.

[0018] Depending on the desired purpose, the DNA can be double-stranded DNA or single-stranded DNA (eg, cDNA). According to certain embodiments, the DNA is a DNA with a 3' recessed end.

[0019] A fourth aspect of the present disclosure provides a method for analyzing the expression of multiple mRNAs in cells of a biological sample. The method comprises: (a) Mechanically disrupting cells in biological samples; (b) in the presence of a metal ion-chelator complex (e.g., Cu-Citrate, Cu-NTA, or Cu-IDA), mixing the product of step (a) with a lysis buffer to release the nuclei from the cells; (c) separating the cell nucleus from the product of step (b); (d) a mixture containing methanol and Mg 2+ Fixing the cell nuclei of step (c) with a solution; (e) mixing the product of step (d) with a reverse transcription (RT) buffer, wherein the RT buffer comprises reverse transcriptase, deoxynucleoside triphosphate (DNTP), and a 5-mercaptoethanol (5-mercaptoethanol). dNTPs) and multiple barcoded RT primers; (f) treating the product of step (e) with an RT reaction to generate a first plurality of cDNAs, each cDNA comprising a first unique barcode sequence; (g) the cDNA of step (f) is mixed with a reagent comprising TdT, deoxyribonucleotides and Co 2+ or Mn 2+ and a reaction buffer containing no K + Mg 2+ And Na + ; (h) treating the product of step (g) at 37° C. for 30-120 minutes to produce a plurality of polyadenylated cDNAs; (i) subjecting the product of step (h) to polymerase chain reaction to produce a plurality of double-stranded cDNAs; (j) sequencing the product of step (i) to confirm the quantity of each unique barcode sequence; and (k) Based on the result of step (j), confirm the expression of mRNA.

[0020] According to certain embodiments, in step (i) of the method of the present disclosure, a plurality of double-stranded DNAs are prepared by the following steps: (i-1) mixing the product of step (h) with a PCR buffer containing an oligo(dT) primer, a DNA polymerase, and dNTPs; and (i-2) Perform PCR using the product of step (i-1).

[0021] Optionally, the method further comprises amplifying the product of step (h) before step (j).

[0022] Optionally, the method further comprises the following steps before step (g): (f-1) mixing the first plurality of cDNAs from step (f) with a ligation buffer comprising a DNA ligase and a plurality of adapters; (f-2) treating the mixture of step (f-1) at 37° C. for 30-120 minutes to produce a second plurality of cDNAs, Each cDNA comprises a second specific barcode sequence linked to the 3' end of the first specific barcode sequence.

[0023] Optionally, the method further comprises repeating step (f-1) and step (f-2) at least once before step (g).

[0024] After reading the following embodiments, a person having ordinary knowledge in the technical field to which the present invention belongs will be able to easily understand the basic spirit and other invention objectives of the present invention, as well as the technical means and implementation aspects adopted by the present invention.

Brief Description of the Drawings

[0025] To make the above and other objects, features, advantages and embodiments of the present invention more apparent and understandable, the accompanying drawings are described as follows.

[0026] Figure 1 This is a silver staining photograph according to Example 1 of the present disclosure, which depicts the activity of TdT under specific reaction conditions, wherein a ssDNA oligonucleotide (SEQ ID NO: 10) was treated with a specific reagent in the presence of 2 mM dATP and 0.25 μL of commercially available TdT. Reaction buffer; K + :potassium acetate; Mg 2+ :Magnesium acetate; Co 2 + : Cobalt chloride. The reactions in lanes 3-8 contained 20 mM Tris-acetate at pH 7.9 and 0.1% X-100. Arrows indicate oligonucleotide substrates without tailing.

[0027] Figure 2 This is a silver staining photograph according to Example 2 of the present disclosure, which describes the effect of divalent cations and their mass on the tail addition of the recessed end DNA matrix. The matrix is ​​in the presence of Mg 2+ or Co 2+Cells were cultured in a reaction mixture containing a tailing cofactor. 5,000 cells from fetal liver were cultured in lysate (Lysate+) and lysis buffer (Lysate-) to simulate the presence or absence of background DNA ends. Arrows indicate substrate without tailing.

[0028] Figure 3 This is a silver staining photograph according to Example 3 of the present disclosure, which describes the dNTP, buffer, reducing agent (dithiothreitol (DTT)) and ion / ion concentration (Mg 2+ The arrows indicate oligonucleotide substrates without tail addition.

[0029] Figure 4 This is a silver-stained photograph according to Example 4 of the present disclosure, which illustrates the effects of TdT, substrate, and cofactor on tail addition efficiency. The arrow indicates the oligonucleotide substrate without tail addition.

[0030] Figure 5 The results of the fluorescence measurement according to Example 5 of the present disclosure describe the Zn 2+ (Zn) and Cu 2+ (Cu) for the inhibition of pancreatic nucleases. Lysates consisted of 4 μL of crude mouse pancreas lysate or a control containing lysis buffer alone (-). Lane 2, represented by squares, and lane 1, represented by circles, served as positive and negative controls for nucleases, respectively. **** indicates p < 0.0001 using lane 2 as a reference in one-way analysis of variance with a Dunnett post hoc test.

[0031] Figure 6 Cu is described in Example 6 of the present disclosure 2+ (Triangle), Cu 2+ Add bovine serum albumin (square), and Cu 2+ Optical density was measured at 600 nm with the addition of bovine serum albumin and citric acid as a chelating agent. The buffers used at different pH values ​​were: pH 4.5: HEPES-SO4; pH 5.5: 2-(N-morpholino)ethanesulfonic acid (MES); and pH 7.2: 4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid (N-(2-Hydroxyethyl)piperazine-N'-(2-ethanesulfonic acid) HEPES.

[0032] Figure 7 The results of fluorescence measurement according to Example 7 of the present disclosure describe Cu 2+ (Square) and Cu 2+ Effect of the complex formed with the chelating agent citric acid (circles) on pancreatic nuclease activity. The symbol # indicates precipitation.

[0033] Figure 8 The results of fluorescence measurement according to Example 8 of the present disclosure describe pH, Cu 2+ Formation of complexes with different chelators and the efficacy of chelators in inhibiting pancreatic nucleases. CuN (Cu 2+ -NTA), CuC(Cu 2+ -citric acid) and sodium citrate solutions C, adjusted to pH 4.5 (panel (A)) or 7.2 (panel (B)). Lysates consisted of 1.5 μL of crude mouse pancreas lysate (+) or lysis buffer control (-). **** indicates p < 0.0001 in one-way analysis of variance with Dunnett's post hoc test, using the corresponding nuclease-free sample as a reference.

[0034] Figure 9 is the fluorescence measurement result according to Example 9 of the present disclosure, which describes Cu 2+ -citrate complex in the inhibition of different types of nucleases (including RNase A (50ng), micrococcal nuclease (200 gel units) and benzonase (gold nuclease, 20 units)). CuC: Cu at pH 7.2 2+ -citric acid. **** indicates p < 0.0001 in one-way analysis of variance with Dunnett's post hoc test using the corresponding nuclease-free sample as the reference.

[0035] Figure 10 The fluorescence measurement results of Example 10 of the present disclosure are based on the results of the fluorescence measurement of Example 10, which describes the fluorescence of the chelating agents NTA, citric acid and ethylenediaminetetraacetic acid (EDTA) in the presence of Cu 2+ Inhibitory efficacy against pancreatic nuclease in the presence of β-lactamase. **** indicates p < 0.0001 using lane 2 as a reference, performed by one-way ANOVA and Dunay's post hoc test.

[0036] Figure 11Fluorescence assay results from Example 11 of the present disclosure illustrate the efficacy of Cu-citrate (CuC) and two other inhibitory treatments (DEPC and low pH) for inhibiting pancreatic nucleases. DEPC: diethyl pyrocarbonate; N: neutral buffer; C: low pH buffer (25 mM citric acid and 250 mM sucrose at pH 3); Lysate: 4 μL of crude lysate from mouse pancreas (+) or lysis buffer control (-). Lanes 2 and 1 served as positive (+) and negative (-) nuclease controls, respectively. **** indicates p < 0.0001 in a one-way ANOVA with Dunnett's post hoc test, using Lane 2 as the reference.

[0037] Figure 12 Figure 12 shows the results of reverse transcription quantitative polymerase chain reaction (RT-qPCR) according to Example 12 of the present disclosure, which depicts the protective effect of the metal-chelator complex on nuclear mRNA in the presence of pancreatic nuclease. HEK293 cell nuclei were cultured in the presence (+) or absence (-) of the complex, at pH 4.5 (squares) or pH 7.2 (circles), and RT-qPCR was performed to assess the enrichment of two housekeeping genes (GAPDH and PSMB4) relative to the genomic DNA control group (dashed line). *, **, and *** indicate p < 0.05, p < 0.01, and p < 0.001, respectively, based on Student's T-test.

[0038] Figure 13 Figure 13 is a box plot depicting the number of genes detected per cell in a resampled 20,000 reads using different techniques in accordance with Example 13 of the present disclosure. The four methods used in Example 13 that achieved high cell capacities comparable to USPPAR are labeled "High," while the other methods that provided lower cell capacities in each experiment are labeled "Low."

[0039] Figure 14 This is a violin plot drawn according to Example 14 of the present disclosure, illustrating the gene detection efficiency of B cells generated using the snRNA sequencing method of the present invention and a commercially available scRNA sequencing platform (10x_v3). 2,500 reads were resampled for each barcode in each cell for plotting. P-values ​​were derived using the Wilcoxon rank order test.

[0040] Figure 15This figure, based on Example 15 of the present disclosure, depicts the gene detection efficiency of maize sprout nuclei using snRNA sequencing with USPPAR and 10xChromium (10x_v3). The median number of reads per nucleus (solid line), UMIs (dashed line), and gene count (dotted line) are plotted at downsampled sequencing depths of 500, 1,000, 2,500, 5,000, and 10,000. All barcodes containing more than 400 genes were retained as the USPPAR dataset. As with the original reference dataset, only high-quality barcodes containing more than 500 genes and with cell type annotations were retained as the reference dataset. [Specific implementation method]

[0041] To provide a more detailed and complete description of the present disclosure, the following provides illustrative descriptions of various embodiments and examples of the present invention; however, these descriptions are not intended to be the only ways to implement or use the embodiments of the present invention. The detailed descriptions cover features of various embodiments and the method steps and sequences for constructing and operating these embodiments. However, other embodiments may also be used to achieve the same or equivalent functionality and step sequences.

[0042] I. Definition

[0043] For convenience, specific proper nouns used in this specification, the embodiments and the appended claims are collected here. Unless otherwise defined in this specification, the meanings of scientific and technical terms used herein are the same as those understood and used by persons of ordinary skill in the art to which the invention belongs. Furthermore, singular nouns used in this specification encompass plural forms of the nouns, and plural nouns used also encompass singular forms of the nouns, unless inconsistent with the context. Specifically, in this specification and the claims, the singular forms "a" and "an" include plural reference values, unless otherwise indicated by the context. In addition, in this specification and the claims, the expressions "at least one" and "one or more" have the same meaning, both representing one, two, three or more.

[0044] Although the numerical ranges and parameters used to define the broader scope of the present invention are approximate, the relevant numerical values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual testing methods. Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1% or 5% of a specific value or range. Alternatively, the word "about" means that the actual value falls within the acceptable standard error of the mean, which is within the acceptable standard error of the mean, as known in the art to which the present invention belongs. It is determined by the consideration of ordinary knowledge. Except for the experimental examples, or unless otherwise explicitly stated, it is understood that all ranges, quantities, values ​​and percentages used herein (for example, to describe the amount of material used, the length of time, temperature, operating conditions, quantitative ratios and other similar ones) are modified by "about". Therefore, unless otherwise stated to the contrary, the numerical parameters disclosed in this specification and the accompanying patent application scope are approximate values ​​and can be changed as needed. At least these numerical parameters should be understood as the number of significant digits indicated and the values ​​obtained by applying the general rounding method. Here, the numerical range is expressed as from one end point to another point or between two end points; unless otherwise stated, the numerical ranges described here include the end points.

[0045] In the present disclosure, a "complex" refers to an aggregate or aggregate of two or more molecules in direct and / or indirect contact with each other. In the present disclosure, a "metal ion-chelate complex" refers to a substance formed by a metal ion and a chelate linked by a covalent bond.

[0046] As used herein, a "chelator" refers to a molecule capable of attaching or binding to a metal ion by forming multiple bonds therewith. Depending on the intended purpose, suitable chelators for forming metal ion-chelator complexes of the present disclosure can be biomolecules (e.g., heme, transferrin, lactoferrin, conalbumin, or ferritin) or organic molecules (e.g., EDTA, citric acid, NTA, IDA, diethylenetriamine, ethylenediamine, N,N',N"-tris(2-pyridylmethyl)-1,3,5-cis,cis-triaminocyclohexane (tachpyr), or other molecules recognizable to one of ordinary skill in the art).

[0047] In the present disclosure, the term "nonionic surfactant" refers to a class of surfactants that do not contain ionic groups and will not be free in aqueous solution. For example, nonylphenoxypolyethoxylethanol (also known as "NP-40"), X-100, polyoxyethylene octylphenyl ether polyoxyethylene (polyoxyethylene octylphenyl etherpolyoxyethylene, also known as " CA-720”), octylphenoxy poly(ethyleneoxy)ethanol (also known as “ CA-630”), polyoxyethylene sorbitan monolaurate (also known as “polysorbate 20” or “ 20”), polyoxyethylene sorbitan monooleate (also known as “polysorbate 80” or “ 80”), polyoxyethylene sorbitan trioleate (also known as “polysorbate 85” or “ 85”), polyoxyethylene sorbitan monopalmitate (also known as “polysorbate 40” or “ 40”), polyoxyethylene sorbitan monostearate (also known as “polysorbate 60” or “ 60”), or a combination of the above.

[0048] In the present disclosure, the term "transition metal" refers to an element selected from Groups Ib, IIb, IIIa (including lanthanum), IVa, Va, VIa, VIIa, and VIII of the periodic table that has an incomplete inner electron shell and acts as a transition link between the most and least electropositive elements in a series of elements. In other words, a "transition metal" refers to an element whose atomic number is between 21-30, 39-48, 57-80, or 89-112.

[0049] The term "nucleic acid" refers to a polymer of nucleotides (e.g., natural and unnatural ribonucleosides and deoxyribonucleosides), including DNA, RNA, and their subclasses (e.g., cDNA, mRNA, etc.). Nucleic acids can be single-stranded or double-stranded and generally contain a 5'-3' phosphoester bond. In addition, in some cases, nucleotide analogs may have other linkages, such as linkers, spacers, and tags available in the art. The nucleotides can include naturally occurring bases (adenosine, guanosine, cytosine, uracil, and thymine) as well as unnatural bases, which may have specialized functions, for example, increasing the stability of the nucleic acid duplex, inhibiting enzymatic digestion, or blocking primer extension or chain polymerization.

[0050] In the present disclosure, the term "barcode" refers to a nucleotide sequence that confers an identity to a molecule or a group of molecules with the same characteristics or origin. A barcode can give a unique identity to an individual molecule (and its copies), such as a unique ID (UID) or a unique molecular identifier (UMI). A barcode can give an identity to an entire population of molecules (and its copies) from the same source (e.g., a sample), such as a multiple ID (MID) or a sample ID (SID). A barcode is of sufficient length and contains a sequence with sufficient diversity to identify a sample based on the barcode associated with the sample. For example, a barcode can be 4-40 nucleotides, 5-36 nucleotides, or 6-30 nucleotides in length. Each barcode in the barcode set has a unique nucleotide sequence, i.e., a sequence that is different from any other barcode in the barcode set. In certain embodiments, the method further comprises identifying the sample based on a target polynucleotide generated by binding to the barcode sequence. Barcoding is a well-known technique in the art: for example, see Winzeler et al., Science (1999), 285:901; Kumar et al., Nature Rev. (2001), 2:302; Giaever et al., Proc. Natl. Acad. Sci. USA (2004), 101:793; Eason et al., Proc. Natl. Acad. Sci. USA (2004), 101:11046; and Justus M. Kebschull et al., Nature Methods (2018), 15:871. The foregoing publications are incorporated herein by reference in their entirety.

[0051] In the present disclosure, the "primer" refers to an oligonucleotide, generally having a free hydroxyl group at the 3' end, which can hybridize or bind to a template (e.g., a specific polynucleotide, a target DNA, a target RNA, etc.), and promote the polymerization reaction of a polynucleotide complementary to the template. In addition to the hybrid sequence that recognizes and binds to the template, the primer may further include a non-hybrid sequence that constitutes the tail end of the primer. For example, the barcoded RT primer of the present disclosure includes a hybrid sequence for recognizing and binding to the target sequence of mRNA (e.g., the polyadenylation tail (polyA) of mRNA), and a barcode sequence Linked to the 5' end or 3' end of the hybrid sequence (preferably the 5' end). It should be understood that even if the primer is not completely complementary to the target sequence, it can still hybridize thereto.

[0052] In this disclosure, the term "adapter" refers to a nucleotide sequence that is added to another sequence to introduce additional properties into that sequence. An adaptor can be single-stranded or double-stranded, or can have both single-stranded and double-stranded segments.

[0053] The term "amplifying" a specific sequence refers to a process for producing multiple copies of a target nucleic acid, for example, DNA. Methods for amplifying nucleic acids are well known in the art, such as PCR and qPCR.

[0054] In this disclosure, "reverse transcription" refers to a reaction in which an RNA template is reversely transcribed into cDNA using a reverse transcriptase. A reverse transcription reaction typically includes an RNA template, a reverse transcriptase, a reaction buffer (e.g., Tris or Tris-HCl), salts, primers (e.g., oligo(dT) primers or random primers), dNTPs, a reducing agent (optionally, such as DTT), and an RNase inhibitor (optionally). As previously mentioned, the mixture can be a complete or incomplete reverse transcription reaction mixture.

[0055] II. Implementation Methods

[0056] The present disclosure is based on the following findings: (1) combining Cu 2+ ions and appropriate chelating agents (i.e., citrate or NTA) can protect RNA from degradation and will not cause undesirable protein aggregation in nuclease-rich samples; (2) containing Mg 2+ Methanol can serve as a stabilizer for non-aggregated nuclei; and (3) Co 2+ or Mn 2+ Based on the above findings, the present disclosure provides a method for quantitatively analyzing and / or confirming the expression profile of single-cell RNA (scRNA) in biological samples by isolating and stabilizing single-cell RNA from biological samples. A tag tail (eg, a barcode sequence) is then added to each single nucleus in the cells of the sample, and the expression profile is quantitatively analyzed and / or confirmed based on the tag tail.

[0057] (i) Methods for inhibiting nuclease activity

[0058] A first aspect of the present disclosure relates to a method for inhibiting nuclease activity in a biological sample. According to certain embodiments of the present disclosure, the method comprises mixing the biological sample with a metal ion-chelator complex to inhibit the activity of the nuclease in the biological sample.

[0059] Depending on the desired purpose, the biological sample can be any sample containing a nuclease, for example, a sample from animal tissue (e.g., spleen tissue, liver tissue, pancreatic tissue, bone marrow, or cells derived from / isolated from the above tissues) or plant tissue (e.g., corn, or cells derived from / isolated from the above tissues).

[0060] In one embodiment of the present disclosure, the nuclease is DNase. In another embodiment of the present disclosure, the nuclease is RNase.

[0061] According to certain embodiments of the present disclosure, the chelator can be a tridentate chelator, a quasidentate chelator, or a pentadentate chelator, which can bind or associate with three, four, or five metal ions, respectively, by forming coordination bonds. In these embodiments, hexadentate chelators (i.e., chelators that bind or associate with six metal ions, such as EDTA) are unable to inhibit nuclease activity after complexing with metal ions. According to certain exemplary embodiments of the present disclosure, the metal ion-chelator complex is Cu-Citrate, Cu-NTA, or Cu-IDA.

[0062] Preferably, the molar ratio of the metal ion to the chelating agent in the complex is between 1 and 10. For example, the molar ratio of the metal ion to the chelating agent in the complex can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. Specifically, the molar ratio of the metal ion to the chelating agent in the complex is equal to 1, i.e., the molar ratio of the metal ion to the chelating agent in the complex is 1:1.

[0063] According to certain embodiments of the present disclosure, a biological sample and a metal ion-chelator complex are mixed in a buffer (e.g., phosphate buffered saline (PBS), Tris-HCl, or HEPES), wherein the concentration of the metal ion-chelator complex in the buffer is 2-100 mM (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100 mM). In certain embodiments, the buffer contains 10-50 mM Cu-Citrate or Cu-NTA to inhibit nuclease activity. According to one embodiment, the buffer for inhibiting nuclease activity contains 50 mM Cu-Citrate. According to another embodiment, the buffer contains 10 mM Cu-NTA to inhibit nuclease activity.

[0064] (ii) Method for isolating cell nuclei from cells

[0065] A second aspect of the present disclosure is a method for isolating cell nuclei from a biological sample using the metal ion-chelator complex described in paragraph (i) of the present disclosure. The method comprises: (a) Mechanically disrupting cells in biological samples; (b) mixing the product of step (a) with a lysis buffer in the presence of a metal ion-chelator complex, to release the nucleus from the cell; and (c) separating the cell nucleus from the product of step (b) to produce the isolated cell nucleus.

[0066] According to various embodiments of the present disclosure, the biological sample can be a sample derived from animal tissue (e.g., spleen tissue, liver tissue, pancreatic tissue, bone marrow, or cells derived from / isolated from the above tissues) or plant tissue (e.g., corn or cells derived from / isolated from corn).

[0067] In step (a), the biological sample is subjected to physical disruption (e.g., mechanical grinding) to release cellular material. Exemplary physical disruption methods known to those skilled in the art include, but are not limited to, microbead milling (also known as bead milling, which involves mixing microbeads with the biological sample and rapidly stirring the mixture to generate strong shear forces around the cells of the biological sample, ultimately separating them), ultrasonic treatment (i.e., utilizing an ultrasonic homogenizer to generate vibrations that induce cavitation to disrupt cells), grinding (i.e., sandwiching the biological sample between two hard surfaces and sliding the two hard surfaces relative to each other to achieve cell disruption), freezing (e.g., using liquid nitrogen or freeze-thaw cycles to disrupt cells by generating ice crystals), and combinations thereof. According to certain exemplary embodiments of the present disclosure, the biological sample is frozen with liquid nitrogen and then ground with an aluminum pan.

[0068] In step (b), the product of step (a) (i.e., the physically disrupted product) is mixed with a lysis buffer to completely release the cell nuclei from the cells. It is understood that the lysis buffer is a buffer solution used for cell disruption, typically comprising a buffer salt (e.g., Tris-HCl or HEPES) and an ionic salt (e.g., NaCl, CaCl2 and / or MgCl2) to adjust the pH and osmotic pressure of the lysate, and to react with one or more surfactants (e.g., X-100 and / or sodium dodecyl sulfate (SDS) to disrupt cell membranes. According to certain embodiments of the present disclosure, the lysis buffer contains 0.1-3% non-ionic surfactant. In certain embodiments of the present disclosure, the lysis buffer contains polysorbate 20 (polysorbate 20, 20) and nonylphenol polyethoxyethylene ether (nonyl phenoxypolyethoxylethanol, NP-40) as surfactants to disrupt cell membranes. Preferably, the lysis buffer contains 0.01-1% (v / v) polysorbate 20 and 0.01-1% (v / v) NP-40 to disrupt cell membranes while maintaining the integrity of the nuclear membrane and / or cell components. Alternatively, the lysis buffer contains 0.01-1% (v / v) polysorbate 20 and 0.01-1% (v / v) X-100 or CA-630 are used to disrupt cell membranes while maintaining the integrity of the nuclear membrane and / or components.

[0069] Preferably, the lysis buffer further comprises Cu-Citrite (IUPAC name: dicopper; 2-hydroxypropane-1,2,3-tricarboxylic acid, also known as cuprocitrol, an ionic compound composed of copper and citric acid) or Cu-NTA (IUPAC name: copper; 2-[bis(carboxymethyl)amino]acetic acid; hydrogen ion, also known as copper-nitrilotriacetate, a complex formed by the combination of copper ions and nitrile triacetate) as an RNase inhibitor. According to certain embodiments, the lysis buffer comprises 2-100 mM Cu-Citrate or Cu-NTA to inhibit RNase activity. In certain embodiments, the lysis buffer comprises 10-50 mM Cu-Citrate or Cu-NTA to inhibit RNase activity. According to one embodiment, the lysis buffer for inhibiting RNase activity comprises 50 mM Cu-Citrate. According to another embodiment, the lysis buffer contains 10 mM Cu-NTA to inhibit RNase.

[0070] In step (c), the nuclei are isolated from the product of step (b). Depending on the desired objective, the nuclei can be isolated / fractionated by FACS screening, using a concentration gradient of iodixanol or a modified sucrose gradient, or any commercially available kit for isolating nuclei. The methods and procedures for isolating / fractionating nuclei are well known to those skilled in the art; therefore, for the sake of brevity, a detailed description thereof is omitted herein. According to certain exemplary embodiments, the nuclei are isolated / fractionated using an iodixanol gradient. In these embodiments, the product of step (b) is layered in a buffer comprising iodixanol (at a low concentration, e.g., 30%) and copper-citrate, with iodixanol (at a high concentration, e.g., 60%) at the bottom. After centrifugation, the nuclei are collected from the interface between the two iodixanol layers (e.g., the interface between 30% and 60% iodixanol).

[0071] Optionally, the method further comprises fixing the cell nuclei separated / isolated in step (c) with an anti-clumping agent (step (d)). As the name implies, an anti-clumping agent can prevent the aggregation and aggregation of cell nuclei. According to an embodiment of the present disclosure, the anti-clumping agent is a Mg-containing 2+ According to certain embodiments, the methanol comprises MgCl2, that is, methanol / MgCl2. In certain exemplary embodiments, Mg 2 The ion is present in methanol at a concentration of 5 mM.

[0072] (iii) Method of adding deoxyribonucleotides to DNA

[0073] A third aspect of the present disclosure relates to a method for adding deoxyribonucleotides to the 3' end of DNA to extend or tail the DNA. The method comprises: (a) mixing DNA with a reaction buffer containing terminal transferase, deoxyribonucleotides, and transition metal ions, wherein the reaction buffer does not contain ions other than the aforementioned transition metal ions (e.g., does not contain K + ions, Mg 2+ Ions and Na + ions); and (b) Incubating the mixture of step (a) at 37° C. for 30-120 minutes to produce DNA having the aforementioned deoxyribonucleotide added to its 3′ end.

[0074] According to various embodiments of the disclosure, the DNA can be ssDNA or dsDNA. In one embodiment, the DNA is cDNA. In another embodiment, the DNA is dsDNA with a 3' recessed end (5' protruding end).

[0075] In step (a), DNA is reacted with a mixture comprising terminal transferase (an enzyme known to catalyze the addition of nucleotides to the 3' hydroxyl terminus of DNA) and deoxyribonucleotides (e.g., deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), and / or deoxyuridine triphosphate (dUTP)). According to certain exemplary embodiments, the deoxyribonucleotide is dATP, and the concentration of dATP in the reaction buffer is between 0.5 mM and 10 mM. In one embodiment, the concentration of dATP in the reaction buffer is 2 mM.

[0076] According to certain embodiments of the present disclosure, the terminal transferase is TdT. In these embodiments, the reaction buffer is TdT buffer (Tris-acetate).

[0077] The reaction buffer of the method of the present disclosure is characterized by having only Co 2+ or Mn 2+ ions, does not contain other ions (including K + Mg 2+ And Na + ions), that is, only Co 2+ or Mn 2+ .

[0078] In step (b), the mixture of DNA, terminal transferase and deoxyribonucleotides is incubated at 37° C. for a period of time (e.g., 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, Preferably, the mixture is incubated at 37° C. for 60-120 minutes to allow the addition of deoxyribonucleotides to the 3′ end of the DNA.

[0079] According to certain embodiments of the present disclosure, in step (a), DNA is mixed with TdT and dATP, and then incubated at 37° C. for 60 minutes to add a homopolymeric tail (polyadenylation (polyA) sequence) to the 3′ end of the DNA.

[0080] (iv) Methods for analyzing mRNA profiles of biological samples

[0081] A fourth aspect of the present disclosure relates to a method for analyzing mRNA activity in cells in a biological sample. The method comprises: (a) Mechanically disrupting cells in biological samples; (b) mixing the product of step (a) with a lysis buffer comprising a metal ion-chelator complex of the present disclosure to release the cell nucleus from the cell; (c) separating the cell nucleus from the product of step (b); (d) containing methanol and magnesium ions (Mg 2+ ) solution to fix the cell nuclei of step (c); (e) mixing the product of step (d) with an RT buffer containing reverse transcriptase, dNTPs, and a plurality of barcoded RT primers; (f) performing an RT reaction on the product of step (e) to generate a first plurality of cDNAs, each cDNA comprising a first specific barcode sequence; (g) the plurality of cDNAs from step (f) are reacted with a terminal transferase (e.g., TdT), dATP, and Co 2+ or Mn 2+ and a reaction buffer containing no K + Mg 2+ And Na + ion; (h) incubating the product of step (g) at 37° C. for a period of time to produce a plurality of polyadenylated cDNAs (i.e., cDNAs having poly(A) tails); (i) performing PCR on the product of step (h) to generate a plurality of double-stranded cDNAs; (j) sequencing the product of step (i) to confirm the quantity of each unique barcode sequence; and (k) Confirming the mRNA expression profile based on the results of step (j).

[0082] Steps (a) to (d) of the method of the present disclosure are similar to the separation method described in paragraph (i) of this specification. Therefore, for the sake of brevity, their detailed description is omitted here.

[0083] In steps (e) and (f), a plurality of cDNAs are generated from the single cell nucleus isolated in step (d), wherein each of the plurality of cDNAs contains a specific tag. Specifically, the single cell nucleus is mixed with an RT reagent (step (e)), and then the mixture is subjected to an RT reaction (step (f)). As is well known in the art, RT reagents provide the necessary components for reverse transcribing mRNA into cDNA; to achieve this purpose, RT reagents generally include reverse transcriptase, dNTPs, RT buffer (e.g., Tris or Tris-HCl), and a plurality of RT primers. According to certain embodiments of the present disclosure, each RT primer is a barcoded RT primer, the structure of which includes a sequence complementary to the target and a barcode sequence. As the name implies, the target complementary sequence includes a nucleotide sequence that is complementary to the target mRNA sequence in the isolated cell nucleus and has binding affinity, for example, an oligo (dT) sequence that recognizes and binds to the poly (A) tail of the mRNA. The barcode sequence may have a length of at least 4 nucleotides (e.g., 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more nucleotides). According to an exemplary embodiment, the barcode sequence of each RT primer has a length of 4 nucleotides. According to another exemplary embodiment, the barcode sequence of each RT primer is 8 nucleotides in length. According to another exemplary embodiment, the barcode sequence of each RT primer is 16 nucleotides in length. In this case, the barcode sequence of the RT primer can be added to the cDNA during synthesis, thereby serving as a tag to identify the cDNA. Based on this unique tag, once the tagged cDNAs are mixed together, the tags can be used to identify the single cell from which each cDNA originated. Therefore, each cDNA can correspond to a single cell.

[0084] The concentration of RT reagents and the procedures for performing RT reactions are well known to those skilled in the art and are omitted for brevity.

[0085] Alternatively, the tagged cDNAs of step (f) (i.e., cDNAs with the first specific tag at the 3' end) are each modified to add another tag (i.e., a second specific tag) to form a diversified tag. According to certain alternative embodiments, the tagged cDNAs of step (f) are mixed with a DNA ligation buffer containing a DNA ligase and a plurality of adapters, wherein each adapter has a second specific tag sequence, i.e., a unique barcode sequence that is different from any other barcode in the adapter set (step (f-1)). Then, the mixture is incubated at 37°C for a period of time (eg, 30-120 minutes) to add the second specific barcode sequence to the 3' end of the first specific barcode sequence (step (f-2)).

[0086] Depending on the desired purpose, steps (f-1) and (f-2) may be optionally repeated at least once, for example, 1, 2, 3, 4 or more times.

[0087] Steps (g) and (h) of the method of the present disclosure are similar to the method described in paragraph (ii) of the present disclosure and are not further described in detail for the sake of brevity.

[0088] In step (i), the polyadenylated cDNA prepared in step (h) (i.e., the first strand) is used as a template to synthesize its complementary strand (i.e., the second strand complementary to the first strand) by PCR. The steps and reaction conditions for preparing dsDNA from cDNA by PCR are well known to those skilled in the art; for the sake of brevity, further details are not described here. According to certain embodiments of the present disclosure, PCR is performed by mixing the cDNA from step (h) with a DNA polymerase, an oligo(dT) primer, dNTPs (including dATP, dTTP, dCTP, and dGTP), and a reaction buffer (e.g., Tris-HCl). Depending on the desired purpose, the reaction buffer may contain one or more salts (e.g., MgCl2).

[0089] Optionally, the dsDNA generated in step (i) is further used to amplify the polyadenylated cDNA carrying the tag by PCR, ie, to increase the copy number of the polyadenylated cDNA carrying the tag.

[0090] Optionally, the PCR product is purified by an appropriate method to separate the tagged polyadenylated cDNA from the PCR reagents (i.e., primers and dNTPs), for example, column purification, colloid purification, ethanol precipitation, polyethylene glycol (PEG) precipitation, magnetic bead purification, etc. According to certain exemplary embodiments of the present disclosure, the PCR product is mixed with magnetic beads in the presence of a PEG / NaCl solution, and the mixture is then placed on a magnetic rack.

[0091] Alternatively, the PCR products are enzymatically digested (eg, using transposase) or mechanically sheared (eg, using ultrasound, focused sonic shearing, hydrodynamic shearing, or nebulization to generate shear force) into DNA fragments for DNA sequencing.

[0092] In step (j), the nucleotide sequence of the dsDNA is confirmed by sequencing, for example, next generation sequencing (NGS) or Sanger sequencing.

[0093] Next, in step (k), the RNA profile is confirmed based on the sequencing results from step (j). Specifically, as described above, barcode tags play an important role in identifying individual cDNAs, their corresponding mRNAs, and the individual cells from which the mRNA / cDNA originated. Therefore, the expression level of each mRNA can be determined by the number of corresponding specific barcode sequences.

[0094] The following working examples are provided to illustrate certain aspects of the present invention to facilitate practice by those skilled in the art. These examples should not be construed as limiting the scope of the present invention. It is believed that a person skilled in the art, after reading the description provided herein, will be able to fully utilize and practice the present invention without undue interpretation. All publications cited herein are incorporated herein by reference in their entirety.

[0095] Example

[0096] Materials and Methods

[0097] Preparation of nucleic acids for barcoding

[0098] A total of 26 nucleic acids were synthesized in this disclosure. The nucleotide sequences of these nucleic acids are summarized in Table 1 below.

[0099] Table 1 Nucleotide sequences of specific nucleic acids

[0100] Example 1: Extension of ssDNA matrix with TdT does not require the monovalent cations and Mg used in current reaction systems. 2+

[0101] 0.5 pmole of ssDNA oligonucleotide (SEQ ID NO: 1) and different components were added to a 10 μL reaction volume and incubated with dATP (2 mM) and commercially available TdT (0.25 μL) at 37°C, 45°C, 55°C, 65°C, and 75°C for 30, 5, 5, 5, and 20 minutes, respectively. All reactions were subjected to electrophoresis analysis ( Figure 1 ).

[0102] In conventional embodiments, TdT is required to utilize a monovalent cation K + ThermoPol buffer, but it is not conducive to tail addition ( Figure 1 , lanes 3 and 4 versus lanes 5 and 6). In addition, the enzyme cofactor in the buffer was Mg 2+ , TdT is not a preferred choice, especially for blunt-ended or recessed-ended substrates (2), such as many cDNA ends found on mRNA. 2+ The tails generated by extending a single-stranded substrate in a buffer containing Co 2+ The tail generated in the buffer is shorter ( Figure 1 , lanes 2 and 8 are compared with lane 7), and Co 2+ Can replace Mg 2+To perform tailing reaction on ssDNA ( Figure 1 , lane 7 vs. lane 8).

[0103] As described above, these results indicate that the cations commonly used in TdT reactions are not conducive to the addition of poly(dA) tails to ssDNA substrates, whereas the use of Co alone 2+ This is sufficient to achieve the goal and can achieve the highest efficiency compared to other reaction systems.

[0104] Example 2: The novel non-ionic formulation enables TdT to achieve 100% efficiency in extending the 3' recessed end of the dsDNA matrix, a result that cannot be achieved by any prior art.

[0105] dsDNA with 3' recessed ends was prepared by binding nucleotide sequences of sequence numbers 2 and 3 at 25 mM concentration in Tris-HCl (25 mM), NaCl (12.5 mM) and EDTA (0.25 mM) at pH 8. 0.25 μmol of the bound substrate was incubated at 37°C for 60 minutes and then at 75°C for 20 minutes in a 20 μL reaction mixture containing PMSF (0.5 mM), dATP (2 mM), X-100 (0.1%), Tris-acetate (20 mM) pH 7.9, and Mg 2+ or Co 2+ In the form of chloride salt (M 2+ The cofactor was added as a tail (at a concentration of 0.75 mM) in the presence (+) or absence (-) of TdT. All reactions were then analyzed by electrophoresis.

[0106] Here, the results show that Co 2+ Replace Mg 2+ The tail addition result of the concave end matrix can be improved. 2+ Replace Mg 2 When the matrix is ​​almost completely up-shifted, it can be confirmed ( Figure 2 , lane 8 versus lane 4).

[0107] The reaction system of the present invention can achieve nearly 100% tail addition at the dsDNA end type that is least susceptible to tail addition, which distinguishes it from any existing method. 2+ The data disclosed this efficacy, previously used 2+ The results also confirmed that different formulations of Co could not completely extend the 3' recessed end. 2+It is not enough to achieve tail addition, but the lack of other inhibitory ions is the key. In addition, it can effectively extend ssDNA (with accessible 3' overhangs, such as Figure 1 ) and 3' recessed dsDNA (with the most inaccessible ends, as Figure 2 ) shows that the reaction system of the present disclosure can completely extend the DNA ends, which is an effect that cannot be achieved by existing reaction systems.

[0108] Example 3 Full tail addition requires the combination of Co 2+ or Mn 2+ With dATP, but does not require dGTP

[0109] As described in Example 2, 5 pmoles of the 3'-recessed substrate was incubated in a 10 μL reaction volume with HEPES (H) or Tris-acetate (T) (20 mM) at pH 7.9, dATP (dA) or dGTP (dG) (2 mM), CoCl2 (Co) or MnCl2 (Mn) (0.75 or 2 mM), DTT (0 or 0.2 mM), and TdT (0 or 0.25 μL) at 37°C for 60 minutes, and then incubated at 75°C for 20 minutes. Finally, half of the reaction volume was analyzed by electrophoresis ( Figure 3 ).

[0110] In addition to dATP and Co 2+ The optimal buffer for the tail addition is reported to be two alternative components, Mn 2+ and dGTP did not improve the tail addition efficiency ( Figure 3 , lane 4 vs. lane 3), Or even unfavorable for the reaction ( Figure 3 , lanes 5 and 6 vs. lanes 3 and 4). In addition, DTT, reducing agent ( Figure 3 , lane 7 vs. lane 6), or using Tris-HCl instead of HEPES as an alternative buffer ( Figure 3 , lane 1 vs. lane 3) It is clear that neither can enhance the tail addition.

[0111] The data demonstrate that the disclosed formulations are flexible with respect to both types of transition metals (but not alkaline earth metals) and the pH-controlled buffers used (e.g., HEPES and TRIS). The use of the reducing agent DTT is optional and does not affect the reaction outcome. However, the choice of dNTP (dATP over dGTP) is crucial for achieving full elongation.

[0112] Example 4: Sufficient amount of TdT is required for complete tail addition.

[0113] Prepare various concentrations (pmole) of 3' recessed end matrix as described in Example 2 in a 10 μL reaction volume and add PMSF (0.5 mM), Tris-acetate (20 mM) at pH 7.9, X-100 (0.1%), dATP (2 mM), CoCl2 (0.75 or 2 mM) and commercially available TdT (0, 0.25 or 1 μL) with (+) or without (-) 5000-cell lysis buffer (Lysate) were incubated at 37°C for 60 minutes, then incubated at 75°C for 20 minutes, and finally all the reactants were analyzed by electrophoresis ( Figure 4 ).

[0114] The final formulation (pH 7.9 concentration of 20mM Tris-acetate, 2mM dATP, 0.75mM CoCl2, 0.1% X-100) and different amounts of TdT (20U / μL), 5 units of TdT can effectively add 2.5pmole of concave end matrix ( Figure 2 , lane 4). However, when the substrate concentration was increased ( Figure 4 , lanes 1-4) or lysates containing 5000-cell cDNA were incubated in the reaction ( Figure 2 , lane 2 vs lane 4), the efficiency of tail addition decreased significantly. The aforementioned problem of decreased tail addition efficiency due to increased substrate mass can be solved by increasing the substrate mass TdT concentration rather than by increasing the cofactor Co 2+ To solve ( Figure 4 , lane 6 versus lanes 7 and 8, respectively).

[0115] In summary, the TdT reaction system based on the optimal component types and concentrations can completely tail the recessed DNA, which was previously unattainable. In addition, the simple reaction formula allows the product to be directly used for other procedures, for example, residual Co can be simply removed by chelation. 2+ PCR amplification can be performed.

[0116] Example 5Cu 2+ Inhibit nuclease activity in pancreatic lysate

[0117] The cells were lysed in 400 μL of lysis buffer (pH 7.2, 20 mM HEPES, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, A crude pancreatic lysate was prepared by lysing 10 mg of mouse pancreas in a solution of 1% dextrose, 0.1% NP-40, and 0.01% digitonin. Lysis was performed by bead beating (10 seconds at 4500 rpm) in a 2 mL plastic tube containing six 2.5 mm diameter magnetic beads. The lysate was centrifuged at 17,000 RCF for 1 minute, and the nuclease-containing supernatant was retained for this assay.

[0118] In a 25 μL reaction volume, an RNaseAlert was used, which is a DNA-RNA-DNA chimera with a green fluorescent molecule at one end and a quencher (50 nM) at the other end. TM Oligonucleotides were added to detect nuclease activity. The reaction was performed in a final reaction volume of 25 μL pancreatic lysis buffer. The specific additives (pancreatic lysis buffer, CuSO4 or ZnSO4) were as follows: Figure 5 After incubation at room temperature for 30 minutes, the reaction was diluted with 150 μL of the same buffer without the substrate and lysis buffer. Fluorescence detection was performed with excitation at 490 nm and emission at 520 nm in triplicate.

[0119] In this example, it is obvious that Cu 2+ According to its concentration, it can effectively inhibit the nuclease activity in pancreatic lysate ( Figure 5 ).

[0120] Example 6 In the presence of proteins such as albumin, Cu 2+ The precipitation problem is more significant, making it unsuitable for biological and clinical applications

[0121] Reactions were prepared in a 96-well plate at room temperature with a total reaction volume of 180 μL per well. Figure 6 As shown, the reactions contained different buffers with specific pH values ​​and specific concentrations of Cu in the form of sulfate (no pH adjustment) or citrate (defined as pH 4.5 or 7.2). 2+ and bovine serum albumin (final concentration 10 μg / μL). The above reaction was repeated three times and the images were taken directly ( Figure 6 , bottom), and measured by optical density at a wavelength of 600 nm ( Figure 6 , upper part) to show the degree of precipitation.

[0122] Hydroxylated Cu 2+ At neutral pH, it is only slightly soluble in water even at concentrations below 1 mM ( Figure 6 , lanes 4-6, marked with triangles). In addition, by 2+Co-culture with bovine serum albumin (BSA) showed that Cu 2+ At pH greater than 5.5, it causes severe protein aggregation, forming a precipitate visible to the naked eye and as determined by OD600 readings ( Figure 6 , lanes 3-6, marked with squares). The above precipitation reaction resulted in the use of Cu alone. 2+ The situation is not suitable for biological and clinical applications because most samples contain proteins and have a neutral pH. 2+ Pre-complexation with citric acid (a chelating agent) resolved the precipitation problem in the presence of BSA at various pH values ​​tested ( Figure 6 , indicated by a circle).

[0123] Example 7 Chelated Cu 2+ With unchelated Cu 2+ Similar efficacy in inhibiting nuclease activity

[0124] Prepare RNaseAlert in a final volume of 25 μL pancreas buffer. TM Oligonucleotide (50 nM), wherein the buffer comprises Figure 7 Specific additives as indicated (pancreatic lysate, CuSO4 or different concentrations of Cu 2+ After incubation at room temperature for 30 minutes, the reaction was diluted to 150 μL with the same buffer without the substrate and lysis buffer, and the fluorescence quantitative assay was performed in triplicate with 50 μL each.

[0125] The above experiments indicate that chelated Cu 2+ It can still block pancreatic nuclease activity in a dose-dependent manner, and its efficacy is comparable to that of unchelated Cu 2+ Similar or even better ( Figure 7 , circle vs. square). This result indicates Chelating agents maintain the beneficial effect of nuclease inhibition while avoiding precipitation issues, making the complex suitable for use in biological systems.

[0126] Example 8: The chelating agent itself cannot inhibit nucleases, but the complex formed can effectively inhibit nucleases at either acidic or neutral pH.

[0127] Prepare RNaseAlert in a final volume of 25 μL pancreas buffer. TM Oligonucleotide (50 nM), wherein the buffer is HEPES-SO4 at pH 4.5 or HEPES at pH 7.2, and contains Figure 8 Specific supplements as indicated (pancreatic lysate, Cu 2+After incubation at room temperature for 30 minutes, 150 μL of the reaction mixture was diluted with the same buffer without the substrate and lysis buffer, and three replicates of 50 μL each were used for fluorescence quantitative analysis.

[0128] The experimental results show that the effect of blocking nuclease is based on Cu 2+ , rather than based on the use of chelating agents alone ( Figure 8 , pH 4.5 and 7.2, lane 5 versus lanes 3 and 4). In addition, Cu 2+ -citric acid complex has inhibitory activity at both acidic and neutral pH ( Figure 8 , pH 4.5 and 7.2, lane 4 vs. lane 2).

[0129] Example 9Cu 2+ - Chelating agent complex is a broad-spectrum nuclease inhibitor that inhibits both DNase and RNase

[0130] Prepare RNaseAlert in a final volume of 25 μL pancreas buffer. TM Oligonucleotide (50 nM), wherein the buffer comprises Figure 9 Specific supplements as indicated (pancreatic lysate, Cu 2+ -citric acid or various nucleases). After incubation at room temperature for 10 minutes, the reaction was diluted to 150 μL with the same buffer without the substrate and lysis buffer, and the fluorescence quantitative assay was performed in triplicate with 50 μL each.

[0131] The experimental results show that Cu 2+ -citric acid complex in acidic and neutral pH environments ( Figure 9 , pH 4.5 and 7.2, lane 4 vs. lane 2) have the ability to block non-selective DNA / RNA endonucleases, for example, they can inhibit the activity of micrococcal nuclease and benzonase, and also inhibit the activity of RNase A ( Figure 9 , pH 4.5 and 7.2, lanes 4, 6, and 8 versus lanes 3, 5, and 7). This result indicates that it can broadly inhibit the activities of different types of nucleases including DNase and RNase.

[0132] Example 10: Occupancy of the coordination site by the chelating agent is the key to determining the inhibitory effect of nucleic acid plum

[0133] Prepare RNaseAlert in a final volume of 25 μL pancreas buffer. TM Oligonucleotide (50 nM), wherein the buffer comprises Figure 10After incubation at room temperature for 30 minutes with the indicated additives (pancreatic lysate, 2 mM CuSO4, or 2 mM of various chelating agents), the reaction was diluted to 150 μL with the same buffer without the substrate and lysate, and three replicates of 50 μL each were performed for fluorescence quantification.

[0134] The data showed that Cu 2+ It can still inhibit nuclease activity when present with citric acid and NTA ( Figure 10 , lane 5; 3D, pH 4.5 and 7.2, lane 3). However, Cu 2+ When present together with EDTA, it cannot effectively inhibit the activity of nuclease ( Figure 10 , lane 6). Cu 2+ The demand for different coordination molecules indicates that Cu 2+ The key to the nuclease blocking effect of the chelator complex lies in the availability of free coordination sites (2 / 6 for citrate and NTA, 0 / 6 for EDTA), rather than the type of chelator.

[0135] Example 11Cu 2+ - Chelator complexes provide nuclease inhibition comparable to the best existing inhibition methods while avoiding their known drawbacks

[0136] Prepare RNaseAlert in a final volume of 25 μL of pancreas buffer (buffer P) or low-pH buffer (buffer C, composed of 25 mM citric acid and 250 mM sucrose at pH 3). TM Oligonucleotide (50 nM), wherein the buffer comprises Figure 11 Specific additives as indicated (pancreatic lysate, 1% DEPC and specific concentrations of Cu 2+ After incubation at room temperature for 30 minutes, the reaction was diluted to 150 μL with the same buffer without the substrate and lysis buffer, and the fluorescence quantitative assay was performed in triplicate with 50 μL each.

[0137] At the optimal concentration of 10 mM ( Figure 11 , lane 3), the complex provides RNase inhibition efficacy equivalent to that of low-pH buffer ( Figure 11 , lane 5), and can achieve an inhibitory effect comparable to that of DEPC ( Figure 11 , lane 4). Finally, compared with the immediate addition of Cu 2+ - Chelating agent complex ( Figure 11 , lane 6), delayed Cu 2+ -Chelator complexes are added at a time point that minimizes fluorescence quenching ( Figure 11, lane 7 vs. lane 2). This result further supports that the complex reduces fluorescence by avoiding cleavage of the DNA / RNA linking the fluorophore and the quencher, rather than directly quenching the fluorophore.

[0138] As described above, the metal-chelator complex exhibits highly effective nuclease blocking efficacy without the drawbacks of low pH-induced protein denaturation, nuclear deformation, or nuclear depurination, as well as DEPC-induced protein and nucleotide modifications that hinder further applications (e.g., immobilization).

[0139] Example 12Cu 2+ -Chelator complexes inhibit nuclease-directed nuclear mRNA degradation at both acidic and neutral pH

[0140] To simulate the extraction of nuclei by direct lysis of the target organ, HEK293 cells were cultured in a nuclease-enriched pancreatic lysis buffer in the presence or absence of Cu 2+ Lysis was performed without the presence of a chelator complex. 30 μL of pancreatic lysis buffer was prepared on ice from a 250x stock solution (pH 4.5 and 7.2, respectively), which contained 50 mM HEPES at pH 4.5 or 7.2, 1x Halt protease inhibitor, 2 μL of pancreatic lysis buffer, and 0 mM (control) or 10 mM Cu 2+ -citrate complex. Add 2 μL of PBS containing 20,000 HEK293T cells and rotate in the refrigerator for 15 minutes to release the cell nuclei. Centrifuge at 1,000 g for 1 minute using a swinging bucket rotor and wash twice with the same buffer (without HALT and with 0.05% polyvinyl alcohol (PVA) replacing the interface active agent) to obtain a cell nucleus pellet. The washed cell nuclei are then lysed at 37°C in 15 μL of lysis buffer (containing 0.2% TX-100 and 200 μg / μL proteinase K) at 1,200 rpm for 30 minutes. The resulting lysate is lysed. The lysate was aliquoted into 20 μL RT reactions, each containing RNA lysis buffer, 0.2 μL PMSF (50 mM in DMSO), 1x RT buffer (50 mM Tris-HCl, pH 8.3; 75 mM KCl; 3 mM MgCl2; 5 mM DTT), dNTPs (final concentration of each dNTP was 0.5 mM), homemade rat RNase inhibitor (100 ng), PVA (0.05%), T23VN (1 μM), and 0 (for genomic DNA control) or 100 ng of homemade reverse transcriptase. The reactions were treated at 42°C for 20 minutes, followed by treatment at 85°C for 5 minutes. All generated cDNAs were used as input for triplicate qPCR reactions. The target genes were the housekeeping genes GAPDH (nucleotide sequences of SEQ ID NOs: 21 and 22), PSMB4 (nucleotide sequences of SEQ ID NOs: 23 and 24), and a genomic DNA fragment (nucleotide sequences of SEQ ID NOs: 25 and 26). The reaction volume for each reaction was 20 μL.

[0141] Compared with the one without Cu 2+ -nuclear samples of citrate complex, Cu 2+ -Citrate complex is essential for protecting and maintaining nuclear RNA from complete degradation by pancreatic nucleases at pH 4.5 and 7.2 ( Figure 12 , containing (+) or not containing (-) CuC). This experimental result confirms that Cu 2+ -citrate complex is suitable for extracting cell nuclei for snRNA-sequencing even in samples rich in nucleases.

[0142] Example 13: Compared to cells prepared using similar RT and barcoding strategies, scRNA-sequencing using the cDNA tailing / amplification system (Examples 1-4) significantly improves the sensitivity of gene detection

[0143] Preparation of cells for USPPAR (Uniform System with Pooled Barcoding and Optimal Polydeoxyadenylation for scRNA Detection)

[0144] After dissociation, the cultured cells were neutralized with an equal volume of PBS-PVA (PBS containing 0.1% PVA), centrifuged at 3,000 RCF for 5 minutes, and then washed with an equal volume of PBS-PVA. Finally, the pellet was resuspended in 20-40 μL of PBS, fixed with 10 volumes of methanol at -20°C, and permeabilized / fixed at -20°C for at least 60 minutes for subsequent use.

[0145] First pooling barcoding based on RT reaction

[0146] The desired number of nuclei was dissolved in methanol, and PVA was added to a final concentration of 0.1%. The methanol was removed by centrifugation at 1,000 g for 1 minute. The pellet was then washed twice with 200 μL of low-salt buffer (10 mM Tris-HCl, pH 8, 1 mM KCl, 0.1% PVA) containing recombinant rat RNase inhibitor (5 ng / μL). The nuclei were resuspended in the same buffer and aliquoted into a 96-well PCR plate for the first barcoding reaction using RT (4,000 cells per 20 μL). The RT reagent consisted of 50 mM Tris-HCl (pH 8.3), 75 mM KCl, 3 mM MgCl2, 5 mM DTT, 0.5 mM of each dNTP, 5 ng / μL homemade rat RNase inhibitor, 0.05% PVA, 7.5% PEG8K, 5 ng / μL reverse transcriptase, 1 μM barcoded T16V primer (SEQ ID NO: 4), and 1 μM barcoded N9 primer (SEQ ID NO: 5). The reaction was performed by rotating the plate at 42°C for 40 minutes, and 0.3 μL of 0.5 M EDTA was added to each well to stop the reaction.

[0147] Subsequent ligation-based pooling and barcoding

[0148] The terminated reaction was pooled into 2-4 microtubes and centrifuged. The tubes were washed with 200 μL of low-salt buffer containing 0.1% PVA and 0.5 ng / μL rat RNase inhibitor, then centrifuged at 1,000 RCF for 1 minute. The wash and centrifugation steps were repeated once. The pellet obtained from the previous step was resuspended in wash buffer and aliquoted into 20 μL portions for subsequent ligation reactions (70 mM Tris-HCl, pH 7.5, 10 mM MgCl₂, 50 mM DTT, 1 mM ATP, 0.1% PVA, 5 ng / μL rat RNase inhibitor, 4 ng / μL T4 DNA, and 1 μM of the barcoding adapter prepared by ligating nucleotides 6 and 7 in the first ligation and the barcoding adapter prepared by ligating nucleotides 8 and 9 in the second ligation using the conditions described in Example 2).

[0149] The reaction was performed by rotating the entire 96-well plate at 37°C for 40 minutes and stopped by adding 0.5 μL of 0.5 M EDTA to each well. Optionally, the ligation reaction was repeated one, two, or three times to diversify the barcode sequences. After the final round of ligation, the terminated reactions were pooled and filtered through an 800-mesh nylon membrane. The cells were washed once with 200 μL of low-salt buffer containing 0.1% PVA and rat RNase inhibitor (0.5 ng / μL) and centrifuged at 1,000 RCF for 1 minute. The pellet was resuspended in an appropriate amount of wash buffer and aliquoted (10,000-20,000 cells per PCR tube in 3 μL) for long-term storage at -80°C.

[0150] cDNA single-tube amplification using the complete tail addition system established in Examples 2-4

[0151] Add 8 μL of To lyse the cells, 3 μL of low-salt buffer containing 1% TdT (275 μg / mL) and 0.275% Tris-100 (0.275%) was added to the lysate. The lysis reaction was performed at 55°C with shaking at 1,500 rpm for 50 minutes. 2 μL of 10x TdT buffer (200 mM Tris-acetate, pH 7.9, 1% TdT) was added to each lysate. The cells were then incubated with 1 μL of DMSO (X-100), 2 μL of dATP (20 mM), 2 μL of CoCl2 (7.5 mM), 0.2 μL of PMSF (50 mM in DMSO), commercially available TdT (2 μL contains 40 units, sufficient for 10,000 cells), and water to a final volume of 20 μL. The cells were then incubated at 37°C for 60 minutes, followed by polydeoxyadenylation at 42°C for 10 minutes, and finally heat-inactivated at 75°C for 20 minutes. After completion of the reaction, 1 μL of EDTA (16 mM) was added to generate residue-free, fully extended cDNA.

[0152] Next, the reaction was added to 58 μL of a mixture containing 16 μL of DNA polymerase buffer (5x), 1.6 μL of dNTP mix (10 mM each), and 8 μL of supT25V (SEQ ID NO: 13, 2 μM). The mixture was divided into two 39 μL aliquots and heated at 94°C for 1 minute, followed by a hold at 45°C. 1 μL of DNA polymerase was added to each reaction. Second strand synthesis was then performed for 54 cycles, with each cycle increasing the reaction temperature by 0.5°C for 20 seconds, followed by a 10-minute reaction at 72°C, and finally a hold at 4°C.

[0153] The reaction mixture was added to 80 μL of a mixture containing 16 μL of DNA polymerase buffer (5x), 1.6 μL of dNTP mix (10 mM each), 16 μL of inhibitor primer (SEQ ID NO: 14, 5 μM), 8 μL of DMSO, and 2 μL of DNA polymerase. The reaction was heat-treated at 98°C for 1 minute, followed by 12 cycles of 98°C for 10 seconds, 68°C for 20 seconds, and 72°C for 4 minutes. EDTA was added to the PCR reaction to a final concentration of 3 mM to terminate enzyme activity.

[0154] Purification of PCR products

[0155] To purify the pre-amplified PCR product, 1 μL of carboxylated paramagnetic beads and a suspension consisting of Tris-HCl (10 mM), PEG8000 (20%), NaCl (2.5 M), EDTA (1 mM), and A PEG / NaCl solution consisting of 0.20% (0.05%) PEG was added to the solution to achieve a final PEG concentration of 7%. The mixture was incubated for 10 minutes and then placed on a magnetic rack. After removing the supernatant, the magnetic beads were washed twice with 500 μL of freshly prepared 80% ethanol-water by inverting the reaction tube 10 times on the magnetic rack, and the supernatant was removed. The beads were left on the magnetic rack for 5 minutes to evaporate any residual alcohol, and then eluted with 8 μL of loTE (10 mM Tris-HCl, pH 8, 0.1 mM EDTA).

[0156] The fragmented cDNA was size-selected using a similar procedure, except that 5.5% PEG was first used to remove high molecular weight DNA. The supernatant was removed from the DNA-coated magnetic beads, and 1 μL of paramagnetic beads and PEG / NaCl solution was added to a final PEG concentration of 7%. This was followed by incubation and washing, and finally 8 μL of PEG / NaCl solution containing 20 (0.1%) loTE elution.

[0157] Fragmentation of pre-amplified DNA by Tn5 digestion

[0158] by DNA extracted from the first-cycle PEG / NaCl purification was detected using a Green dye and calibrated using a calibration curve generated with known plasmid DNA concentrations. 50 ng of preamplified DNA and 50 ng of vector plasmid were fragmented with 0.5 μL of 2 μM Tn5 at 55°C for 30 minutes. Tn5 was loaded with ligation adapters containing the i5 PCR handle (prepared by ligating SEQ ID NOs. 15 and 16 under the conditions described in Example 2) in 10 μL of fragmentation buffer (10 mM Tris-HCl, pH 8.5, 5 mM MgCl2, 10% DMF). Tn5 was inactivated by adding 2.5 μL of quenching buffer (0.25% SDS, 21 mM EDTA) and heating at 55°C for 10 minutes. 100 ng of plasmid DNA was used for experimental fragmentation to determine the optimal amount of Tn5, followed by inactivation reaction and gel electrophoresis analysis to obtain an intensity peak at the 800 bp target. 1 μL of the quenched reaction was used to confirm the optimal cycle number in a 10 μL qPCR reaction, wherein the reaction consisted of DNA polymerase buffer (1x), dNTPs (0.2 mM each), i5 primer (SEQ ID NO: 17, 200 nM), supAGC primer (SEQ ID NO: 18, 200 nM), X-100 (0.1%), Green (1 / 30,000 v / v), ROX (500 nM), and 0.125 μL of DNA polymerase. The PCR thermal cycle consisted of denaturation at 98°C for 1 minute, followed by 20 cycles of 98°C for 10 seconds, 63°C for 15 seconds, and 72°C for 30 seconds. The number of cycles that produced half the maximum fluorescence value was used as the PCR condition for the subsequent fragmented samples. DMSO, water, P7supAGC (SEQ ID NO: 19), and Nextera i5 primers (SEQ ID NO: 20) were substituted. Green, ROX, supAGC primers and i5 primers. Next, 0.5M EDTA was added to a final concentration of 3mM, and the PCR products were size-selected using the aforementioned PEG / NaCl.

[0159] result

[0160] This systematic optimization was performed in USPPAR to collect single-cell transcriptomes from a pool of human HEK and mouse bone marrow MS5 cells, resampling approximately 1,500 cells from 100,000 single-prep cells. Initially, the results showed clear species-specific enrichment of barcoded reads, with only minimal multiplexing in the species pooled experiment (data not shown). The 1,204 cells resampled from a 100,000-cell input showed a low multiplexity of 0% (after 3 cycles of barcoding), and the 16,450 cells resampled from 400,000 cells had a multiplexity of 0.2%, which is comparable to other high-throughput cell-throughput methods using combinatorial barcoding (e.g., 10,000-13,000 cells obtained from a standard 100,000 cells using the SPLiT-Seq kit showed a multiplexity of 1.3-1.7% and 39,400 cells obtained using the PIP-Seq v4 kit had a multiplexity of 3.3%), and significantly lower than the 6.4% multiplexity expected from 16,000 cells using a 10x Chromium HT channel. Notably, the low multiplexity does not increase with increasing the number of resampled libraries in the same experiment, as each library is assigned an additional layer of barcodes during amplification.

[0161] The sequencing results were then benchmarked against reference databases from multiple state-of-the-art platforms, including 10x Chromium (HEK293T and NIH / 3T3 cells), Quartz-sequencing 2 (annotated HEK293 cells), VASA-sequencing (HEK293T and mouse embryonic stem cells (mESCs)), sci-RNA-sequencing (HEK293T, HeLa S3, and NIH / 3T3 cells), PIP-sequencing (HEK293T and NIH / 3T3 cells), and SPLiT-sequencing (HEK293, HeLa S3, and NIH / 3T3 cells). Joint modeling of these databases revealed four clear The defined cell populations—HEK293 cells, HeLa S3 cells, mESCs, and mouse stromal cells—reflect the input cell types of each platform. In addition to the shared populations of cell types in UMAP, pseudopopulation reads from human cells were also separated by cell type. Beyond differentiation by cell type, adjacent regions also exhibited technology-dependent differences: outputs from sci-RNA-seq, SPLiT-seq, VASA-seq, and USPPAR correlated highly with each other, followed by 10x Chromium and Quartz-seq2, and finally PIP-seq. USPPAR's similarity to sci-RNA-seq, SPLiT-seq, and VASA-seq is reflected by their shared high ratio of intronic reads, a property that aids in inferring cell-state transitions. To demonstrate the informativeness of intronic reads, mouse liver is used as an example, as the cellular resolution of a mixture of HEK293 and MS5 cells is insufficient. In the liver, independent modeling using intron count alone yielded distinct populations reflecting the correct cell type. This result indicates that reads aligning to intronic regions are informative and originate from transcripts rather than from untranscribed genomic DNA.

[0162] To benchmark detection sensitivity across technologies, reads from HEK293 cells were independently filtered for each platform to display the UMIs and genes detected in each cell for the same readout. Figure 13 , genes), and the cumulative number of genes detected from increasing numbers of randomly sampled cells. These evaluations showed that the sensitivity of USPPAR-based detection was equal to or comparable to that of sorting- or microfluidics-based methods (10x Chromium, VASA-sequencing, and Quartz-sequencing2), and surpassed that of other high-throughput combinatorial barcode-based methods (SPLiT-sequencing, sci-RNA-sequencing, and PIP-sequencing). Considering the possibility of reduced sensitivity due to expression intensity, USPPAR showed a low dropout rate similar to that of specific instrument-based methods and had a higher number of genes that passed quality filtering compared to the other three combinatorial barcode-based methods (USPPAR had 13,271 genes, sci-RNA-sequencing, SPLiT-sequencing, and PIP-sequencing had 11,937, 11,302, and 10,433 genes, respectively).

[0163] Based on the complete DNA tailing reaction system of the first invention aspect of this disclosure, USPPAR offers higher gene detection efficiency than current high-throughput methods. USPPAR offers scalable processing capabilities of at least 100,000 to 400,000 cells, surpassing other high-sensitivity methods that rely on specialized equipment. Furthermore, USPPAR exhibits low multiplexity and dropout rates, and exhibits good gene expression correlation with other platforms.

[0164] Notably, compared to SPLiT-sequencing (a similar system for cell barcoding, but differing in that SPLiT-sequencing utilizes a template exchange approach to amplify the cDNA tail), USPPAR detected a significantly higher number of genes. This result further demonstrates that using the complete tail addition system of the present invention for DNA amplification can significantly enhance gene detection efficiency.

[0165] Example 14 Combining Optimized cDNA Tailing (Examples 1-4) and Nuclease Inhibition (Examples 5-12) to Achieve a One-Pot SnRNA-seq Method for Comprehensive Understanding of Spleen Cell Status

[0166] One-pot extraction of nuclei from nuclease-rich mouse spleen cells

[0167] Mouse spleens were immediately frozen in liquid nitrogen until ready for use. Tissue fragments were placed in folded polyimide films, frozen in liquid nitrogen for at least 10 minutes, and crushed in liquid nitrogen with an aluminum block on a stainless steel plate to maintain a low temperature. The resulting powder was immediately lysed with 1,000 μL of ice-cold lysis buffer (pH 4.5, 20 mM HEPES, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2), wherein the lysis buffer contained a surfactant mixture (0.1% 20, 0.1% NP-40 and 0.01% digitonin) and Cu 2+ -Citric acid (diluted to 10 mM from a 250 mM stock solution at pH 4.5). The mixture was ground in a microcentrifuge tube with a plastic pestle and filtered through an 800 mesh nylon membrane. The filtrate was then layered onto 500 μL of the same lysis buffer (containing 10 mM Cu 2+-citric acid and 0.05% PVA to replace the surfactant mixture, 30% iodixanol), and 20 μL of 60% iodixanol was placed at the bottom as a cushion. After centrifugation at 2,000 g for 5 minutes, the nuclei were collected from the 30%-60% iodixanol interface and washed with 200 μL of washing buffer (pH 4.5, concentration 20 mM HEPES, 146 mM NaCl, 1 mM CaCl2, 21 mM MgCl2, 0.05% PVA, 10 mM Cu 2+ -citric acid) were washed twice on a column with a hydrophilic PTFE membrane (pore size 3 μm). The nuclei were resuspended and fixed with 200 μL of wash buffer containing 0.125% paraformaldehyde at 4°C for 15 minutes. 10 μL of Tris-HCl (pH 8, 1 M) was added to quench the paraformaldehyde. The nuclei were then fixed with 10 volumes of methanol (containing a final concentration of 5 mM MgCl2) at -20°C and stored at -20°C until further use. The subsequent snRNA sequencing procedure was identical to the scRNA sequencing procedure described in Example 13, with the only difference being that in this example, the nuclei were washed on the PTFE membrane during the pooling and barcoding process.

[0168] Since the lysis solution contains Cu 2+A one-pot lysis of mouse spleen (an organ rich in nucleases and blood) and snRNA-sequencing analysis was achieved by combining a chelating agent complex, double fixation with PFA and methanol, and gentle cell washing using a filter membrane. This snRNA-sequencing method improves the barcoding loss of lymphocytes previously observed using a low-pH lysis buffer system. Furthermore, by using the same number of barcodes per Leiden cluster and maintaining a consistent number of reads per barcode (2,500) for normalization, the optimized snRNA-sequencing system demonstrated an increase in the number of genes detected per barcode. Furthermore, a purified splenocyte library obtained using the 10x commercial platform (sample ID: 10k_Mouse_Splenocytes_5p_gemx, published on April 16, 2024) was used for additional benchmarking. The snRNA-sequencing results of the present disclosure integrated well with commercial databases, demonstrating that the proportion of blood cell types was comparable to that identified by the commercial 10x platform, which requires selective enrichment of spleen cells (USPPAR vs. 10x_v3). Notably, the one-pot nuclear extraction of the present disclosure demonstrated a greater than 10-fold increase in the expression of interstitial or endothelial-specific markers in Col11a1 and Cdh5 cells, respectively. Based on a normalized comparison using the cell type with the most barcodes (B cells), the system of the present disclosure detected more genes than the commercial 10x platform when the analyzed cells had the same number of resampled reads (Figure 14, USPPAR vs. 10x_v3). In summary, the one-pot lysis system of the present disclosure provides a proportion of blood cell types comparable to the 10x platform, increased coverage of interstitial cells, and high gene detection sensitivity, demonstrating its ability to efficiently address challenging organs in a convenient manner.

[0169] In summary, unlike previous methods that require sequencing incomplete subsets of individual cells from challenging organs (e.g., the spleen), the disclosed method for nuclease inhibition effectively and completely captures the original cellular state of nuclease-rich organs. Compared to the state-of-the-art 10xChromium system, the disclosed method improves sensitivity through a unique combination of a broad-spectrum nuclease inhibitor and a comprehensive cDNA tailing system.

[0170] Example 15 The combination of RNA protection and DNA amplification is also suitable for one-pot lysis of plant tissues to improve the sensitivity of gene detection in snRNA-sequencing compared to the most advanced commercial platforms (Examples 1-12)

[0171] One-pot extraction of nuclei from nuclease-rich mouse spleen cells

[0172] Corn seeds were placed on wet paper towels for 4 days to germinate. The bud fragments were frozen, broken and lysed in the same manner as the spleen fragments, except that the bud fragments were lysed using a Ca-free 2+ The cells were lysed in a lysis buffer (pH 4.5, 20 mM HEPES, 146 mM NaCl, 21 mM MgCl2 and 0.5 mM EGTA), wherein the lysis buffer contained Cu 2+ - Citric acid (diluted to 10 mM from a 250 mM stock solution at pH 4.5) and a surfactant mixture. The cells were further homogenized 20 times with Dounce to release the nuclei and filtered through a nylon membrane with a size of 800 mesh. The filtrate was then layered onto 500 μL of the same lysis buffer (containing 10 mM Cu 2+ -citric acid and 0.05% PVA to replace the surfactant mixture, 30% iodixanol), and 20 μL of 60% iodixanol was placed at the bottom as a cushion. After centrifugation at 2,000 g for 5 minutes, the nuclei were collected from the 30%-60% iodixanol interface and washed with 200 μL of washing buffer (pH 4.5, concentration 20 mM HEPES, 146 mM NaCl, 21 mM MgCl2, 0.5 mM EGTA, 0.05% PVA, 10 mM Cu 2+ -citric acid) were washed twice by centrifugation at 250 g for 1 minute, followed by washing with 20 μL of 10 mM Cu 2+ The cells were resuspended in the same buffer containing citric acid and fixed with 10 volumes of methanol containing 5 mM MgCl2 for at least 30 minutes at -20°C. The subsequent snRNA-seq procedure was identical to the scRNA-seq procedure described in Example 13, with the only difference being that in this example, the nuclei were washed by centrifugation at 250 g for 1 minute during the pooling and barcoding process to reduce residual large organelles.

[0173] Compared to a reference nucleus database prepared using the 10x platform, nuclei prepared using USPPAR had more UMIs and genes detected across all resampled reads per barcode (Figure 15, USPPAR vs. 10x_v3). Furthermore, the multiplexity was very low (0.2%) when nuclei from three different species were prepared simultaneously. Furthermore, the bud database could be modeled and co-embedded with the reference data (USPPAR vs. 10x_v3; data not shown). The inferred Leiden assignments remained well-populated when transferred to a 2D UMAP generated using the USPPAR dataset alone, which was modeled and embedded. Finally, the informativeness of the data is further supported by the matching marker expression between the reference database and the USPPAR database, for example, Leiden3 has the mesophyll cell marker Zm00001eb158810, Leiden11 has the bundle sheath cell marker Zm00001eb033390, and Leiden14 has the protoxylem marker Zm00001eb076470.

[0174] It should be understood that the foregoing description of the embodiments is given by way of example only, and that various modifications may be made by those having ordinary knowledge in the technical field to which this invention belongs. The above description, examples, and experimental results provide a complete description of the structure and use of exemplary embodiments of the present invention. Although various specific embodiments of the present invention are disclosed in the above embodiments, they are not intended to limit the present invention. Those having ordinary knowledge in the technical field to which the present invention belongs may make various changes and modifications thereto without departing from the principles and spirit of the present invention. Therefore, the scope of protection of the present invention shall be based on the scope defined by the accompanying patent application.

Claims

1. A method for inhibiting nuclease activity in a biological sample, comprising mixing the biological sample with a metal ion-chelator complex to inhibit the activity of the nuclease in the biological sample.

2. The method of claim 1, wherein the metal ion-chelating agent complex is copper citrate, copper nitrilotriacetate or copper iminodiacetate.

3. The method of claim 1, wherein the molar ratio of metal ion to chelating agent in the metal ion-chelating agent complex is between 1 and 10. 4 . The method of claim 3 , wherein the molar ratio of the metal ion to the chelating agent in the metal ion-chelating agent complex is equal to 1.

5. A method for isolating cell nuclei from cells in a biological sample, comprising: (a) mechanically disrupting cells in the biological sample; (b) mixing the product of step (a) with a lysis buffer in the presence of a metal ion-chelator complex to release the nuclei from the cells; and (c) separating the cell nucleus from the product of step (b) to produce the isolated cell nucleus.

6. The method of claim 5, wherein in step (c), the cell nucleus is isolated by: (c-1) treating the product of step (b) by concentration gradient centrifugation in the presence of the metal ion-chelating agent complex; and (c-2) collecting a fraction containing the cell nucleus from the product of step (c-1).

7. The method of claim 5, wherein the metal ion-chelating agent complex is copper citrate, copper nitrilotriacetate or copper iminodiacetate.

8. The method of claim 5, wherein the molar ratio of metal ion to chelating agent in the metal ion-chelating agent complex is between 1 and 10. 9 . The method of claim 8 , wherein the molar ratio of the metal ion to the chelating agent in the metal ion-chelating agent complex is equal to 1.

10. The method of claim 5, wherein the lysis buffer in step (b) comprises 0.1-3% (v / v) of a non-ionic surfactant.

11. The method of claim 5, wherein the concentration of the metal ion-chelator complex in the lysis buffer is about 2-100 mM.

12. The method of claim 5, further comprising: (d) fixing the cell nuclei obtained in step (c) with a solution comprising methanol and magnesium ions.

13. The method of claim 12, wherein the concentration of the magnesium ions in the solution is about 5 mM.

14. A method for adding deoxyribonucleotides to the 3' end of deoxyribonucleic acid (DNA), comprising: (a) mixing the DNA with a reaction buffer, wherein the reaction buffer comprises terminal deoxynucleotidyl transferase (TdT), the deoxyribonucleotides, and a transition metal ion, wherein the reaction buffer does not contain ions other than the transition metal ion; (b) incubating the mixture of step (a) at 37° C. for 30-120 minutes to prepare the DNA having the deoxyribonucleotide at the 3′ end. The method according to claim 14 , wherein the transition metal ion is a cobalt ion or a manganese ion.

16. The method of claim 14, wherein the DNA is a complementary deoxyribonucleic acid (cDNA). The method of claim 14 , wherein the DNA is a double-stranded DNA having a 3′ recessed end.

18. The method of claim 14, wherein the deoxyribonucleotide is deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), deoxythymidine triphosphate (dTTP), or deoxyuridine triphosphate (dUTP). The method according to claim 18 , wherein the deoxyribonucleotide is dATP, and the concentration of dATP in the reaction buffer is 0.5-10 mM.

20. The method of claim 19, wherein the concentration of dATP in the reaction buffer is 2 mM.

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