A sc3T-seq method for accurate analysis of APA sites in single cells

Abstract

The present invention discloses a sc3T-seq method for accurately analyzing APA sites in single cells, which relates to the fields of molecular biology and bioinformatics. The method comprises incubation of oligo(dT)20 primers with streptavidin magnetic beads, addition of a small amount of starting sample and exogenous single-stranded DNA, synthesis of double-stranded cDNA, breakage of the double-stranded cDNA, removal of polyA, release of labeled cDNA, addition of biotin-labeled λ-DNA, addition of carboxyl magnetic beads, screening and removal of exogenous cDNA, end repair, addition of linkers, construction of a cDNA library, and removal of biotin-labeled λDNA with streptavidin magnetic beads to obtain a purified sample library. The present invention reduces the amount of starting sample, making the experimental technique for analyzing APA sites applicable to a small amount of samples or even single cells, and has a great effect on the accurate detection and analysis of cell-specific APAs.

Description

Technical Field

[0001] The present invention relates to the fields of molecular biology and bioinformatics, and in particular to an sc3T-seq method for accurately analyzing APA sites in single cells on a genome-wide scale. Background Art

[0002] APA is a key post-transcriptional regulatory event, involved in the fundamental regulatory mechanism of gene expression in various cancers. Differential expression of 3'UTR isoforms in different cancer cell lines suggests a strong link between APA and cancer. APA events alter mRNA coding sequences or 3'UTR length, directly or indirectly influencing a range of important biological processes, including embryonic development, cell proliferation and differentiation, immune response, neuronal activation, and tumorigenesis. Studies have shown that APA is tissue- and cell-type-specific and is also influenced by environmental factors (Chen, W., et al.; Gruber, A.J. and M. Zavolan.).

[0003] With the continuous improvement and accumulation of gene expression data in public databases, genome-wide analysis of APAs has become possible through bioinformatics analysis of expressed sequence tags (ESTs). Global APA variations can also be analyzed using microarray methods and paired-end ditag (PET) analysis. With the development of high-throughput sequencing technology and advances in bioinformatics, APAs in sequences have been analyzed using RNA-seq. However, for APA research, only reads covering the 3' end of mRNA are useful, and these only account for a small portion of RNA-seq data. RNA-seq coverage of the 5' and 3' ends of genes is relatively low overall, making RNA-seq unsuitable for accurate and comprehensive determination of polyadenylation sites.

[0004] Studies have found that 3'-enriched RNA sequencing (3'-enriched RNA-seq) can better identify polyadenylation sites than standard RNA-seq methods and better quantify mRNAs with 3'UTRs of varying lengths. Although these technologies can effectively capture the 3' end of mRNA, most, such as PAS-seq and Poly(A)-seq, struggle to avoid the effects of the Poly A structure on sequencing during library construction. Xu Anlong (Fu, Y., et al.) developed the SAPAS technology, which introduces artificial mutations into the primers during PCR, incorporating base C into the Poly T sequence to reduce sequencing signal confusion caused by the Poly structure during high-throughput sequencing. However, this method also has its limitations, such as the fact that heating the RNA to break it can also damage the Poly A structure, leading to partial RNA loss.

[0005] The oligo dT-based method developed by Calvin H. Jan et al. (Jan CH., et al.), Poly(A) Position Profiling by Sequencing (3P-seq), constructs a 3'UTR-tagged library at the RNA stage. This library is then sequenced after reverse transcription to confirm that the library tags are derived from RNA, completely avoiding the internal priming problem. However, this method involves a complex library construction process, and the majority of the experimental procedures are performed at the RNA level, requiring high experimental conditions and techniques. The multiple steps of RNA ligation and enzyme digestion also result in significant RNA loss, making it unsuitable for constructing libraries from small sample sizes.

[0006] Therefore, technicians in this field are committed to developing a 3T-seq method that requires extremely low starting sample amounts and accurately analyzes APA sites in single cells, so that this experimental technology can be applied to a small number of samples or even single cells. Summary of the Invention

[0007] In view of the above-mentioned defects of the prior art, the technical problem to be solved by the present invention is how to reduce the starting sample amount and develop a 3T-seq method that can accurately analyze single-cell APA sites with extremely low starting sample amount, so that this experimental technology can be applied to a small number of samples or even single cells.

[0008] To achieve the above objectives, the present invention provides an sc3T-seq method for accurately analyzing APA sites in single cells, comprising the following steps:

[0009] Step 1: Prepare Gsu I-Oligo(dT)20 magnetic beads by combining magnetic beads with primer probes. Design and synthesize an exogenous single-stranded DNA fragment or single-stranded RNA with poly A, add it to the sample, and then add it to the prepared Gsu I-Oligo(dT)20 magnetic beads. Reverse transcription of mRNA is performed to synthesize first-strand cDNA.

[0010] Step 2: Perform a synthesis reaction between the prepared second-strand synthesis system and the resuspended first-strand cDNA obtained in step 1 to synthesize the second-strand cDNA;

[0011] Step 3: Resuspend the second-strand cDNA obtained in step 2 in a fragmentation enzyme reaction system. Place on ice for 5 minutes, then add 5 μL of double-stranded DNA fragmentase NEB to perform a fragmentation reaction to randomly fragment the newly synthesized cDNA to obtain the fragmented cDNA.

[0012] Step 4: Add the prepared digestion reaction system to the centrifuge tube containing the fragmented cDNA obtained in step 3, resuspend it, and perform enzyme digestion reaction. The 3'UTR end is released from the GsuⅠ-Oligo(dT)20 magnetic beads to obtain the 3' end tag original library;

[0013] Step 5: Add exogenous λ DNA to the 3'-end tagged original library obtained in step 4, purify the cDNA fragments and remove the exogenous single-stranded DNA fragments to obtain a DNA tag library;

[0014] Step 6: The DNA tag library obtained in step 5 is end-repaired to obtain end-modified DNA. The 3' end of the end-modified DNA is tailed with an A tail, connected to an Illumina high-throughput sequencing adapter, PCR amplified with a high-fidelity enzyme, and library fragments are screened. After the library is constructed, the biotin-labeled λ-DNA is removed and the purification system is used to obtain a library free of exogenous DNA contamination.

[0015] Furthermore, the nucleotide sequence of the primer probe is shown in SEQ ID No.1.

[0016] Furthermore, the nucleotide sequence of the exogenous single-stranded DNA fragment in step 1 is shown as SEQ ID No. 2.

[0017] Furthermore, the components of the two-strand synthesis system in step 2 are: 30 μL of lotion 1, 184 μL of enzyme-free water, 25 μL of 10× two-strand synthesis buffer, 5 μL of 10 mM dNTP (+dUTP, -dTTP), 1 μL of DNA ligase, 4 μL of DNA polymerase, and 1 μL of ribonuclease H; wherein the components of the lotion 1 are: 280 μL of enzyme-free water, 80 μL of 5× one-strand synthesis buffer, and 40 μL of 0.1 M dithiothreitol.

[0018] Furthermore, step 2 also includes: taking 50 μg of ribonuclease-free exogenous single-stranded DNA fragments and single cells, mixing them, quick-freezing them at -80°C for more than 2 hours, then heating and denaturing them in a 70°C constant temperature metal bath for 3 minutes, and immediately placing them on ice to cool for 1 minute.

[0019] Furthermore, the components of the fragmentation enzyme reaction system in step 3 are: 39.5 μL of water, 5 μL of 10× fragmentation enzyme reaction buffer, 0.5 μL of 100× bovine serum albumin, and 5 μL of matching cleavage enzyme.

[0020] Furthermore, in step 3, the fragmentation reaction temperature is 37° C. and the reaction time is 22 min.

[0021] Furthermore, the components of the digestion reaction system in step 4 are: 10 μL of 10×buffer B, 2 μL of 0.5 mmol / L S-adenosylmethionine, 2 μL of GsuⅠ, and 86 μL of double-distilled water; wherein the buffer B is a buffer solution matching GsuⅠ, and the concentration of GsuⅠ is 5 U / L.

[0022] Furthermore, the mass of exogenous λDNA added in step 5 was 100 ng; and 0.9x magnetic beads were used to remove exogenous single-stranded DNA fragments.

[0023] Furthermore, in step 6, streptavidin magnetic beads are used to remove biotin-labeled λ-DNA.

[0024] Furthermore, the present invention designs and synthesizes an exogenous single-stranded DNA fragment and adds it to the sample.

[0025] Furthermore, AMPureXP Beads were used to purify the tagged cDNA in subsequent experimental steps to remove exogenous DNA fragments.

[0026] Furthermore, a biotin-labeled λ-DNA (biotin-λ-DNA) was designed and synthesized.

[0027] Furthermore, after the library construction is completed, biotin-λ-DNA is removed using biotin-coated magnetic beads to obtain a library free of exogenous DNA contamination.

[0028] Furthermore, the addition of a special exogenous single-stranded DNA fragment maintains the binding efficiency of the mRNA poly(A) tail to the magnetic beads and the efficiency of the subsequent enzymatic reaction even with low sample amounts. The sequence of this single-stranded exogenous DNA fragment is shown in SEQ ID No. 2: 5'TGGTATGGGGCGGGTAGATGGCTCCAGAACTAGCTCAAAAAAAAAAAAAAAAAAAACTCCAGAACTAGCTC3'. This synthesized exogenous DNA fragment has the following characteristics: 1. It contains two recognition sites for the enzyme Gsu I. When the tag is subsequently released by Gsu I, the exogenous DNA can be enzymatically cleaved into smaller fragments, facilitating the removal of exogenous DNA through fragment screening; 2. The length of this fragment is appropriate to ensure the efficiency of subsequent reverse transcription. The principle behind this is that when the sample concentration is extremely low, the probability of collisional binding between molecules in the solution is reduced, thereby reducing the efficiency of the mRNA poly(A) tail binding to the magnetic beads with the oligo(dT)20 primer. Furthermore, this can also reduce sample loss during the experiment.

[0029] Furthermore, after Gsu I digestion, biotin-labeled λ-DNA was added to AMPureXP Beads before purification, facilitating cDNA purification and library construction. Streptavidin magnetic beads were used to remove the biotin-labeled λ-DNA before library amplification to prevent its impact on subsequent high-throughput sequencing results.

[0030] In preferred embodiment 1 of the present invention, the experimental process and principle of the sc3T-seq method for single-cell APA sites are described in detail;

[0031] In preferred embodiment 2 of the present invention, the specific implementation process of the sc3T-seq method for single-cell APA sites is described in detail.

[0032] The beneficial technical effects of the present invention are as follows:

[0033] The 3T-seq method developed by the present invention combines the advantages of multiple methods to develop a unique technology that uses TT base endings to accurately locate the APA site at the end of the 3' UTR, while also avoiding sequencing disorders caused by poly structures. This technology overcomes the shortcomings of previous technologies in the following aspects: 1. Constructing a special oligo(dT)20 primer that can not only screen for RNA containing poly A but also contains an enzyme cleavage site to remove the poly A structure in the library; 2. Using double-stranded cDNA break enzyme to break the double-stranded cDNA at the DNA stage, reducing RNA loss; 3. Introducing a Gsu I enzyme recognition site at the 5' end of the oligo(dT)20 primer to remove the poly A structure at the DNA level, facilitating operation and reducing experimental difficulty; 4. The TT sticky end left after enzyme cleavage is used to screen data and accurately locate the APA site.

[0034] Compared with 3T-seq, the advantage and innovation of the sc3T-seq of the present invention is that it reduces the starting sample amount, making the experimental technology for analyzing APA sites applicable to a small number of samples or even single cells, which is very useful for the accurate detection and analysis of cell-specific APAs.

[0035] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the experimental process of the sc3T-seq method for single-cell APA sites according to a preferred embodiment 1 of the present invention;

[0037] Figure 2 Schematic diagram of the experimental principle of the existing 3T-seq method according to a preferred embodiment 1 of the present invention;

[0038] Figure 3 This is a microscopic image of a single cell or a small number of cells cultured from the single cell according to a preferred embodiment 2 of the present invention;

[0039] Figure 4 This is a diagram of gel excision and recovery after PCR amplification according to a preferred embodiment 2 of the present invention;

[0040] Figure 5 This is a graph showing the results of the quality inspection at 2100 after the library construction is completed in a preferred embodiment 2 of the present invention. DETAILED DESCRIPTION

[0041] The following describes several preferred embodiments of the present invention with reference to the accompanying drawings to make its technical content clearer and easier to understand. The present invention can be embodied in many different forms of embodiments, and the scope of protection of the present invention is not limited to the embodiments mentioned herein.

[0042] Example 1 Experimental Principle

[0043] The experimental process of the present invention is as follows Figure 1 As shown, oligo(dT)20 primers are incubated with streptavidin magnetic beads, a small amount of starting sample and exogenous single-stranded DNA are added, double-stranded cDNA is synthesized, double-stranded cDNA is broken, poly A is removed, labeled cDNA is released, biotin-labeled λ-DNA is added, carboxyl magnetic beads are added, exogenous cDNA is screened and removed, the ends are repaired, adapters are added, and a cDNA library is constructed. The biotin-labeled λDNA is removed by streptavidin magnetic beads to obtain a pure sample library. The experimental principle of the existing 3T-seq method is as follows Figure 2 shown.

[0044] The present invention overcomes the shortcomings of previous technologies in the following aspects: 1. A special oligo(dT)20 primer is constructed, which can not only screen RNA containing poly A but also contain an enzyme cleavage site to remove the poly A structure in the library; 2. Double-stranded cDNA breakase is used to break the double-stranded cDNA at the DNA stage, reducing the loss of RNA; 3. A Gsu I enzyme recognition site is introduced at the 5' end of the oligo(dT)20 primer to remove the poly A structure at the DNA level, facilitating operation and reducing experimental difficulty; 4. The TT sticky end left after enzyme cleavage is used to screen data and accurately locate the APA site.

[0045] Example 2 sc3T-seq method for single-cell APA sites

[0046] The sc3T-seq method for genome-wide, precise analysis of APA sites in single cells follows:

[0047] Step 1. Biotin-GsuⅠ-Oligo(dT)20 coated magnetic beads

[0048] Binding of magnetic beads to primer probes

[0049] 1.1 Take 50 μl of M280 streptavidin magnetic beads in an enzyme-free tube and place it on a magnetic rack until the solution is completely clear. After aspirating the supernatant, remove the centrifuge tube;

[0050] 1.2 Wash the magnetic beads three times with binding buffer 1, 100 μl each time;

[0051] The recipe for Binding Buffer 1 is as follows:

[0052] Enzyme-free water 10mL 5 mol / L sodium chloride 8mL 0.5 mol / L EDTA 10mL Total 20mL

[0053] 1.3 Take 5 μl of 100 μM oligo (dT) 20 primer and 95 μl of binding buffer 1 and mix them in a magnetic bead tube. Then rotate and mix them on a rotary mixer at room temperature and incubate for 2 hours.

[0054] Step 2. Reverse transcription of mRNA to synthesize first-strand cDNA

[0055] Binding of mRNA poly(A) tail to magnetic beads

[0056] 2.1 Remove the centrifuge tube from the rotary mixer and place it on a magnetic rack. After the solution is clarified, remove the supernatant;

[0057] 2.2 Remove the centrifuge tube, resuspend with 200 μL of binding buffer 1, and then wash once with 200 μL of binding buffer 1;

[0058] 2.3 Wash three times with binding buffer 2, 100 μL each time.

[0059] The recipe for Binding Buffer 2 is as follows:

[0060] 1MTris-HCl (PH7.0) 2mL 8M lithium chloride 1.25mL 0.5M EDTA (pH 8.0) 0.4mL 0.1 M dithiothreitol 1mL Enzyme-free water 15.35mL Total 20mL

[0061] 2.4 Take 50 μg of RNase-free exogenous DNA with poly A and mix it with single cells, freeze it at -80℃ for more than 2 hours, then heat it in a 70℃ constant temperature metal bath for denaturation for 3 minutes, and immediately place it on ice to cool for 1 minute; the single cells or a small number of cells cultured from the single cells during the experiment are shown in the following figure. Figure 3 As shown, taking Jurkat cells as an example, after single cells were cultured for 5, 10, and 15 days, when the cell quantity was small, the cells were collected and RNase-free poly A exogenous DNA was added.

[0062] 2.5 Add Binding Buffer 2 to bring the total volume up to 1 mL, then add to the prepared GsuⅠ-Oligo(dT)20 magnetic beads and incubate at room temperature for 10 minutes. Gently shake to allow the beads to fully bind.

[0063] 2.6 Synthesis of first-strand cDNA

[0064] 2.6.1 Wash the beads twice with detergent A, 500 μL each time.

[0065] The formula of Lotion A is as follows:

[0066] 1MTris-HCl (pH 7.0) 200 μL 8M lithium chloride 37.5μL 0.5M EDTA (pH 8.0) 40 μL 10% sodium lauryl sulfate 211.8μL Glycogen (RNA level) 10 μL Enzyme-free water 19.163mL Total 20mL

[0067] After 2.6.2, wash once more with 500 μL of detergent B.

[0068] The formula of Lotion B is as follows:

[0069] 1MTris-HCl (pH 7.0) 200 μL 8M lithium chloride 37.5μL 0.5M EDTA (pH 8.0) 40 μL Glycogen (RNA level) 10 μL Enzyme-free water 19.375mL Total 20mL

[0070] 2.6.3 Rinse the magnetic beads four times with 100 μL of 1× first-strand synthesis buffer, where the 1× first-strand synthesis buffer is a commercial reagent.

[0071] 2.6.4 Prepare the reverse transcription system in a PCR tube. The reverse transcription system preparation is shown in Table 1.

[0072] Table 1 Reverse transcription system

[0073]

[0074]

[0075] *2.5mM dNTP Mix with methylated dCTP is prepared by mixing 100mM dATP / dGTP / dTTP and 10mM methylated dCTP in the corresponding proportions.

[0076] Reaction conditions:

[0077] 37℃ for 30 minutes

[0078] 42℃ for 30 minutes

[0079] Gently shake the system every 5 minutes to prevent the magnetic beads from settling.

[0080] The reaction system was placed on ice to terminate the reaction.

[0081] Step 3. Synthesis of Second-Strand cDNA

[0082] 3.1 After the reaction is terminated, place the centrifuge tube on a magnetic rack. After the solution is clarified, remove the supernatant;

[0083] 3.2 Wash the magnetic beads with detergent 1 for a total of 3 washes, 100 μL each time; (This step of washing should be performed as quickly as possible because the magnetic beads will be very dry in this washing solution, easily sticking to the tube wall but difficult to wash off, resulting in the loss of magnetic beads)

[0084] The formula of lotion 1 is shown in Table 2.

[0085] Table 2 Lotion 1

[0086] Enzyme-free water 280μL 5× first-strand synthesis buffer 80 μL 0.1 M dithiothreitol 40 μL Total 400 μL

[0087] 3.3 Prepare the second-strand synthesis system on ice:

[0088] The preparation of the two-strand synthesis reaction system is shown in Table 3.

[0089] Table 3 Two-chain synthesis reaction system

[0090] Lotion 1 30 μL Enzyme-free water 184μL 10× Second-chain Synthesis Buffer 25 μL 10mM dNTP (+dUTP, -dTTP) 5μL DNA ligase 1 μL DNA polymerase 4 μL RNase H 1 μL Total 250 μL

[0091] *The dNTPs used here were prepared by mixing 100 mmol / L dATP / dGTP / dCTP / dUTP in the corresponding ratios.

[0092] Mix the prepared second-strand synthesis system with the resuspended magnetic beads. Incubate at 16°C for 2 hours. Check the centrifuge tube every 5 minutes during the reaction and mix gently.

[0093] 3. After 42 hours, place on ice, place on a magnetic rack for clarification, and remove the supernatant to terminate the reaction;

[0094] 3.5 Preheat detergent C to 75°C and dissolve. Wash twice with detergent C, 200 μL each time.

[0095] The formula of Lotion C is as follows:

[0096] 2.5MTris 40 μL 0.5M ethylenediaminedicarboxylic acid (pH 8.0) 20 μL 5M sodium chloride 4mL 10% sodium lauryl sulfate 2mL Glycogen (molecular level) 10 μL Double distilled water 14mL Total 20mL

[0097] 3.6 Wash the beads three times with detergent D, 200 μL each time (to remove SDS in detergent C to prevent it from affecting downstream enzymatic reactions).

[0098] The formula of Lotion D is as follows:

[0099]

[0100] Step 4. Random fragmentation of newly synthesized cDNA

[0101] 4.1 Wash the second-strand cDNA with 2*100μl detergent 2; (to adapt the magnetic beads to the enzyme digestion system)

[0102] The formulation of lotion 2 is shown in Table 4.

[0103] Table 4 Lotion 2

[0104]

[0105]

[0106] 4.2 Prepare the fragmentation enzyme reaction system on ice;

[0107] The reaction system of the fragmentation enzyme has a formula as shown in Table 5.

[0108] Table 5 Reaction system of fragmentation enzyme

[0109] Double distilled water 39.5μL 10× Fragmentase Reaction Buffer 5μL 100× bovine serum fetal bovine albumin (10 mg / mL) 0.5μL Cleavage enzyme (NEB) (matching enzyme, add after 5 minutes on ice) 5μL Total 50 μL

[0110] 4.3 Resuspend the magnetic beads with the fragmentation enzyme reaction system, place on ice for 5 minutes, and then add 5 μL of double-stranded DNA fragmentation enzyme (10,000 U / mL);

[0111] 4.4 Place in a 37°C water bath and react for 22 minutes (the time here must be accurate; if the time is too long, the fragments will become more fragmented, so you can adjust it). During the reaction, remove the centrifuge tube from the water bath every 5 minutes and gently flick the centrifuge tube to mix the system and prevent all the magnetic beads from settling to the bottom of the tube;

[0112] After 4.522 min incubation, the reaction was immediately terminated by placing on ice;

[0113] Step 5. Release of 3'UTR Ends from Magnetic Beads

[0114] Release of target fragment

[0115] 5.1 Take out the centrifuge tube from the ice and insert it into the magnetic rack. Let it stand for a while and remove the supernatant.

[0116] 5.2 Wash the magnetic beads 5 times with detergent 3, 100 μL each time;

[0117] 5.3 After that, wash 4 times with detergent 4, 100 μL each time;

[0118] The formulation of lotion 3 is shown in Table 6.

[0119] Table 6 Lotion 3

[0120]

[0121] The formulation of lotion 4 is shown in Table 7.

[0122] Table 7 Lotion 4

[0123]

[0124] 5.4 Then, prepare the digestion reaction system with GsuⅠ restriction enzyme on ice;

[0125] The digestion reaction system formula of GsuⅠ restriction endonuclease is shown in Table 8.

[0126] Table 8 Digestion reaction system formula of GsuⅠ restriction endonuclease

[0127]

[0128] 5.5 Add the prepared digestion reaction system to the centrifuge tube containing magnetic beads and resuspend it;

[0129] 5.6 Afterwards, place the centrifuge tube in a metal bath for enzyme digestion at 30°C for 2 hours. During the reaction, remove the centrifuge tube and gently tap it every 5 minutes to prevent the magnetic beads from settling to the bottom of the tube.

[0130] After 72 hours, place the centrifuge tube on a magnetic rack and let it stand at room temperature for 2 minutes. After clarification, aspirate the supernatant and transfer it to a new centrifuge tube.

[0131] 5.8 Then add 100 μL of LOTE (10 mM Tris-HCl, 1 mM ethylenediamine dicarboxylic acid, pH 8.0) to the centrifuge tube to further elute the library. After standing on the magnetic rack for a while, aspirate the supernatant and combine it with the previous supernatant to obtain the 3' end tag original library (a total of 200 μL).

[0132] Step 6. Purification of cDNA fragments

[0133] The original library was purified using the phenol-chloroform-isoamyl alcohol extraction method. The specific purification method is as follows:

[0134] 6.1 Add 100 ng of exogenous λ DNA to the upper wash buffer of the original library, then add an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1, pH 8.0), vortex and thoroughly mix until the liquid turns milky white, and centrifuge at 13000 rpm for 5 minutes at room temperature;

[0135] 6.2 After centrifugation, carefully aspirate the upper aqueous phase and transfer it to a new centrifuge tube;

[0136] 6.3 Add 2.5 times the volume of anhydrous ethanol, 0.1 / 0.2 / 0.25 volume of 3 mol / L sodium acetate (pH 5.2), and 0.01 volume of glycogen in sequence, mix thoroughly by inversion, and freeze at -80°C for 30 minutes or at -20°C overnight to aid nucleic acid precipitation.

[0137] 6.4 After sedimentation is complete, centrifuge at 20,000 g for 30 minutes at 4°C and discard the supernatant.

[0138] After 6.5, wash the precipitate twice with 75% ethanol, centrifuge at 20,000 g for 5 min at 4°C, and discard the supernatant;

[0139] 6.6 Use a mini centrifuge to remove the liquid from the tube wall, aspirate it with a pipette, and then dry the precipitate;

[0140] 6.7 Finally, add 35 μL of 10 mmol / L Tris-HCl (pH 8.0) to resuspend and dissolve the precipitate.

[0141] 6.8 Qubit quantification: 198 μL buffer + 1 μL sample, add 1 μL dye in a dark place, mix manually, and let stand in a dark place for 2 minutes.

[0142] Step 7. Removal of foreign DNA

[0143] After Gsu I enzyme treatment, exogenous DNA is reduced to fragments less than 100 bp and removed using 0.9x magnetic bead purification. The reason for not directly replacing phenol precipitation with magnetic bead purification is that the original library volume of 200 μL is too large, requiring a large amount of magnetic beads, which is uneconomical.

[0144] Purified using 0.9×AMPureXP Beads.

[0145] Step 8. High-throughput library construction

[0146] 8.1 Library DNA end repair and 3' end A addition

[0147] The reaction system of the Rapid Terminal Modification Kit is shown in Table 9.

[0148] Table 9 Rapid terminal modification kit reaction system

[0149]

[0150] Reaction conditions: PCR instrument, 20°C, 5 min. (Heated lid closed)

[0151] After the reaction, double-distilled water was added to make up the total volume of the system to 200 μL, and the terminal-modified DNA was recovered by phenol-chloroform extraction and ethanol precipitation and dissolved in 24 μL Tris-HCl.

[0152] The A tail added to the 3' end is shown in Table 10.

[0153] Table 103' with A tail at the end

[0154] Blunt-end DNA tagging library 24 μL 10×ExTaq buffer 3μL 10mM dATP 2.5 μL ExTaq enzyme 0.5μL Total 30 μL

[0155] Reaction conditions: 70°C, 30 min, heated lid 85°C.

[0156] After the reaction, the cells were purified using 0.9×AMPureXP Beads and eluted with 12 μL Tris-HCl.

[0157] 8.2 Connecting to Illumina high-throughput sequencing adapters

[0158] Connect to Illumina high-throughput sequencing adapter p5 / p7 adapter.

[0159] The connection system is shown in Table 11.

[0160] Table 11 Connection system

[0161] End-modified DNA library 12 μL 5×T4 DNA ligation buffer 4 μL 50% PEG4000 2μL P5 / P7 adapter (630 ng / μL) 1 μL T4 DNA ligase (5 U / μL) 1 μL Total 20 μL

[0162] Reaction conditions: 16°C, 12 hours.

[0163] After the reaction, water was added to make up to 50 μL, and the mixture was purified and screened twice with 0.8×AMPureXP Beads, and finally eluted with 8.5 μL Tris-HCl.

[0164] Quibit quantification determines the subsequent PCR cycle.

[0165] 8.3 PCR Amplification with High-Fidelity Enzymes and Library Fragment Screening

[0166] PCR amplification

[0167] This time, the Q5 Mix in the DNA library preparation kit was used, and the system is shown in Table 12.

[0168] Table 12Q5 Mix System

[0169] 3'UTR library 7.5 μL 2×Q5Mix 12.5μL P5 primer (10 μM) 2.5 μL P7 primer (10 μM) 2.5 μL Total 25 μL

[0170] The PCR procedure is as follows:

[0171]

[0172] Figure 2: Gel excision and recovery after PCR amplification Figure 4 As shown. After agarose gel electrophoresis, DNA fragments were enriched in the 200-400 bp range. The theoretical fragment size for this experiment is 250-300 bp. Under ultraviolet light, this agarose segment was cut out for subsequent gel recovery.

[0173] After the library is built, the results of the 2100 quality inspection are as follows: Figure 5 The peak shape of the waveform indicates that the DNA library is normal and can be used for subsequent experiments.

[0174] 8.4 Removal of biotin-λ-DNA and purification system

[0175] 1) Transfer 10 μL of M280 streptavidin-coated magnetic beads (Invitrogen, 11205D) to a PCR tube; add 1 mL of 1x BW buffer and resuspend in a vortex for 5 seconds. Collect the beads on a magnetic rack and resuspend in 10 μL of 1x BW buffer. The formula for 2x BW buffer is as follows:

[0176] 1MTris-HCl (pH 7.5) 150μL 0.5M EDTA 30μL 5M sodium chloride 6ml Double distilled water to 15ml

[0177] 2) Add the prepared samples to the pre-washed magnetic beads and incubate at 30 rpm for 2 h at room temperature.

[0178] 3) Enrich the magnetic beads, collect the supernatant, extract and purify with phenol chloroform, precipitate the DNA with ethanol, and finally dissolve in 20 μl of 10 mM Tris-HCl (pH 8.0).

[0179] The preferred embodiments of the present invention have been described in detail above. It should be understood that numerous modifications and variations based on the concepts of the present invention are possible without inventive effort by those skilled in the art. Therefore, any technical solution that can be derived by one skilled in the art through logical analysis, reasoning, or limited experimentation based on the concepts of the present invention and the prior art should be within the scope of protection defined by the claims.

Claims

1. A sc3T-seq method for accurately analyzing APA sites in single cells, characterized by: The method comprises the following steps: Step 1: Prepare Gsu I-Oligo (dT)20 magnetic beads by combining magnetic beads with primer probes; design and synthesize an exogenous single-stranded DNA fragment, add it to the sample, and then add it to the prepared Gsu I-Oligo (dT)20 magnetic beads, and reverse transcribe the mRNA to synthesize the first-strand cDNA; Step 2: Perform a synthesis reaction between the prepared second-strand synthesis system and the resuspended first-strand cDNA obtained in step 1 to synthesize the second-strand cDNA to obtain double-stranded cDNA; Step 3, Fragmentation Enzyme Reaction System: Resuspend the double-stranded cDNA obtained in step 2, place on ice for 5 min, and then add 5 μL of double-stranded DNA fragmentase NEB to perform a fragmentation reaction to randomly break the newly synthesized double-stranded cDNA to obtain the fragmented cDNA; Step 4: Add the prepared digestion reaction system to the centrifuge tube containing the fragmented cDNA obtained in step 3, resuspend it, and perform enzyme digestion reaction. The 3'UTR end is released from the Gsu I-Oligo (dT)20 magnetic beads to obtain the 3' end tag original library; Step 5: adding exogenous λ DNA to the 3'-end tagged original library obtained in step 4, purifying the cDNA fragments and removing the exogenous single-stranded DNA fragments to obtain a DNA tag library; Step 6: The DNA tag library obtained in step 5 is end-repaired to obtain terminal-modified DNA, the 3' end of the terminal-modified DNA is A-tailed, and the DNA is ligated with a high-throughput sequencing adapter, amplified by PCR with a high-fidelity enzyme, and the library fragments are screened. After the library is constructed, the biotin-labeled λ-DNA is removed and the system is purified to obtain a library free of exogenous DNA contamination; the nucleotide sequence of the primer probe is shown in SEQ ID No. 1; The nucleotide sequence of the exogenous single-stranded DNA fragment in step 1 is shown in SEQ ID No. 2; The components of the second-strand synthesis system in step 2 are: 30 μL of detergent 1, 184 μL of enzyme-free water, 25 μL of 10× second-strand synthesis buffer, 5 μL of 10 mM dNTP, where the dNTP is obtained by mixing 100 mmol / L dATP / dGTP / dCTP / dUTP in corresponding proportions, 1 μL of DNA ligase, 4 μL of DNA polymerase, and 1 μL of ribonuclease H; wherein the components of detergent 1 are: 280 μL of enzyme-free water, 80 μL of 5× first-strand synthesis buffer, and 40 μL of 0.1 M dithiothreitol; the components of the fragmentation enzyme reaction system in step 3 are: 39.5 μL of water, 5 μL of 10× fragmentation reaction buffer, 0.5 μL of 100× bovine serum albumin, and 5 μL of the matching fragmentation enzyme; The components of the digestion reaction system in step 4 are: 10 μL of 10× buffer B, 2 μL of 0.5 mmol / L S-adenosylmethionine, 2 μL of Gsu I, and 86 μL of double-distilled water; wherein the buffer B is a buffer solution for Gsu I, and the concentration of Gsu I is 5 U / L; the mass of the exogenous λ DNA added in step 5 is 100 ng; the exogenous single-stranded DNA fragments are removed using 0.9x magnetic beads; In step 6, streptavidin magnetic beads are used to remove the biotin-labeled λ-DNA.

2. The sc3T-seq method according to claim 1, wherein The step 2 further comprises: taking 50 μg of the exogenous single-stranded DNA fragments free of ribonuclease and mixing with single cells, quick freezing at -80°C for more than 2 hours, then heating and denaturing in a 70°C constant temperature metal bath for 3 minutes, and immediately placing on ice for cooling for 1 minute.

3. The sc3T-seq method according to claim 1, wherein The fragmentation reaction temperature in step 3 is 37° C., and the reaction time is 22 min.

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