Construction method and application of high-throughput single cell transcriptome library based on probe targeted enrichment

Abstract

The invention belongs to the field of single cell level nucleic acid detection, and particularly relates to a construction method and application of a high-throughput single cell transcriptome library based on probe targeted enrichment. According to the method, single cell fixed punching and hybridization enrichment of a specific targeting double probe designed by a target nucleic acid sequence are combined, the nucleic acid sequence comprises mRNA and non-coding RNA, the probe is connected with beads with barcodes through a connecting sequence on the probe, cell labeling is carried out, and library building steps such as extension and amplification are completed; therefore, the problems of insufficient transcriptome integrity, single detection target and low number of detected genes are solved.

Description

Technical Field

[0001] The present invention belongs to the field of nucleic acid detection at the single-cell level, and specifically relates to a high-throughput single-cell transcriptome library construction method and application based on probe targeted enrichment. Background Art

[0002] The transcriptome refers to all RNA transcribed by a cell at a specific stage, including mRNA and non-coding RNA. Some non-coding RNAs lack poly(A) tails and play a crucial role in regulating cellular gene expression. Single-cell transcriptome sequencing technology has developed rapidly in recent years, revealing cellular gene expression patterns, cell subpopulation distribution, and heterogeneity, playing a vital role in fields such as oncology, developmental biology, and neuroscience.

[0003] Current single-cell sequencing technologies require cell lysis followed by reverse transcription of the first-strand cDNA using a polyT primer. The first-strand cDNA is then used as a template for second-strand synthesis via PCR, ultimately resulting in double-stranded cDNA. This not only results in the loss of some non-coding RNA, but also often results in significant RNA loss during the RNA extraction process, limiting the ability to obtain a complete transcriptome, the types of genes identified, and the overall gene detection rate. Summary of the Invention

[0004] One purpose of the present invention is to use fixation reagents to cross-link proteins, maintain cell structure and lock the expression state of nucleic acids, thereby solving the problems of single-cell sample storage and multi-sample batch processing.

[0005] Another object of the present invention is to use a pore-forming reagent to pore the plasma membrane and nuclear membrane while maintaining the cell structure, so that RNA in the cytoplasm and nucleus can be targeted without extracting RNA, thereby achieving the construction of a complete transcriptome.

[0006] Another purpose of the present invention is to design specific targeted dual probes based on target nucleic acid sequences for hybridization enrichment, where the nucleic acid sequences include mRNA and non-coding RNA. The probes are connected to beads with barcodes through the linker sequence on the probes to perform cell labeling and complete library construction steps such as extension and amplification, thereby solving the problems of single detection target and low number of detected genes.

[0007] The technical solution of the present invention: In a first aspect, the present invention provides a method for constructing a high-throughput single-cell transcriptome library based on probe-targeted enrichment, comprising the following steps: (1) Providing one or more cells or cell nuclei, fixing them and performing perforation treatment; The pore punching treatment is performed using a Triton X-100 solution having a volume concentration between 0.1% and 0.3%; (2) contacting the fixed cells or cell nuclei with one or more nucleic acid-specific targeting dual probes, wherein the nucleic acid-specific targeting dual probes comprise a first probe and a second probe, wherein the first probe comprises a 3'-end nucleic acid-specific targeting sequence and a first universal sequence, and the second probe comprises a 5'-end nucleic acid-specific targeting sequence and a first linker sequence, so that the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridize with a first target region and a second target region of a target RNA transcript in the cell or cell nucleus, respectively, and the first target region is adjacent to the second target region; (3) mixing the cells or cell nuclei treated in step (2) with the enzymatic reaction mix to obtain a cell phase, which is then mixed with beads and an oil phase to prepare water-in-oil droplets; The enzymatic reaction mix comprises a ligation reagent that allows the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridized with the target RNA transcript to be ligated together to form an RNA-probe double strand, a reagent that removes RNA from the RNA-probe double strand, and an extension reagent; The beads are connected with a barcode sequence, a second universal sequence and a second connection sequence; (4) performing an enzymatic reaction in the water-in-oil droplet, so that the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridized with the target RNA transcript are linked together to form an RNA-probe double strand, RNA is removed from the RNA-probe double strand, and the first linker sequence is hybridized with the second linker sequence and extended along the probe chain to obtain a barcoded probe; (5) Demulsification is performed to recover the barcoded probes, pre-amplification is performed, and the enriched fragments are labeled using Index PCR. After product purification, a single-cell transcriptome library that can be directly used for sequencing is obtained.

[0008] Furthermore, the fixation treatment is performed using paraformaldehyde with a concentration of 0.5%-8%, preferably 4%-8%.

[0009] Furthermore, the Triton X-100 solution further comprises NaCl, sucrose, HEPES (pH 7.4) and optionally MgCl2 and CaCl2.

[0010] Furthermore, each of the one or more nucleic acid-specific targeting dual probes targets the same or different target nucleic acids.

[0011] As used herein, target nucleic acids include RNA transcripts of cells or nuclei, including mRNA and non-coding RNA.

[0012] Furthermore, the nucleic acid-specific targeting dual probe is a single-stranded DNA probe.

[0013] Furthermore, the first probe and the second probe are 25-50 nt in length.

[0014] Furthermore, the first base at the 5' end of the second probe is A or T.

[0015] Furthermore, the 5' end of the second probe comprises a phosphorylation modification.

[0016] Furthermore, the target RNA transcript includes mRNA and non-coding RNA.

[0017] Furthermore, the ligation reagent that ligates the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridized with the target RNA transcript to form an RNA-probe double strand is SplintR ligase.

[0018] SplintR ligase is a new type of DNA ligase that is only suitable for connecting adjacent single-stranded DNAs fixed by complementary RNA and has extremely high specificity.

[0019] Furthermore, the reagent for removing RNA from the RNA-probe double strand includes RNase H.

[0020] Furthermore, the extension reagent includes dNTPs, DNA polymerase or Mu polymerase.

[0021] Furthermore, the beads are sequentially connected with a second universal sequence, a barcode sequence and a second connection sequence.

[0022] As used herein, the preparation of water-in-oil droplets can be performed using any method and instrument known in the art, as long as single cell encapsulation can be achieved.

[0023] Furthermore, the oil phase is Partitioning Oil.

[0024] Furthermore, the pre-amplification is performed using pre-amplification primers targeting the first universal sequence and the second universal sequence.

[0025] In a second aspect, the present invention provides a high-throughput single-cell transcriptome library constructed by the method described herein.

[0026] In a third aspect, the present invention provides use of the high-throughput single-cell transcriptome library as described herein in single-cell sequencing.

[0027] Beneficial effects of the present invention 1. The sample of the present invention is a cell sample fixed with a reagent, which is convenient for storage and batch processing of samples, and solves the problem of high requirements for sample freshness in the prior art.

[0028] 2. The present invention uses a pore-forming reagent that can simultaneously pore the plasma membrane and nuclear membrane, so that subsequent probe hybridization enrichment can target RNA in the plasma membrane and nuclear membrane. Combined with the highly specific SplintR ligase, it can specifically target and enrich the target RNA, thereby constructing a complete transcriptome.

[0029] 3. The present invention uses single-stranded DNA double probes for nucleic acid sequence enrichment, which has good specificity and high detection sensitivity, and improves the gene detection rate compared with existing single-cell technology.

[0030] 4. The present invention can simultaneously detect mRNA and non-coding RNA at single-cell resolution, enriching the detection targets compared to existing single-cell technologies. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1 A schematic diagram of the probe used in the present invention is shown.

[0032] Figure 2 A schematic flow chart of the construction method of the present invention is shown.

[0033] Figure 3 The quality control results of the transcriptome library prepared by the present invention are shown.

[0034] Figure 4 The sensitivity test results of the construction method of the present invention are shown. DETAILED DESCRIPTION

[0035] The present invention is further described below with reference to specific examples, which, however, are not intended to limit the present invention in any way. Unless otherwise specified, the reagents, methods, and equipment used in the present invention are conventional reagents, methods, and equipment in the art.

[0036] Example 1 This example provides a method for constructing a high-throughput single-cell transcriptome library based on probe-targeted enrichment. The specific steps are as follows: 1. Targeted Probe Design and Synthesis The probe is a single-stranded DNA probe, comprising a first probe and a second probe, which are complementary to the first target region and the second target region adjacent to the nucleic acid sequence, respectively. The length of the first probe and the second probe is 25-50nt. The first probe comprises a first universal sequence and a 3' end targeting sequence. The universal sequence is used for pre-amplification of the probe, and the 3' end target sequence targets the hybrid nucleic acid sequence. The second probe comprises a 5' end targeting sequence and a first linker sequence. The 5' end target sequence targets the hybrid nucleic acid sequence, and the first linker sequence is used to connect to the barcoded beads for cell labeling. The 5' end of the second probe contains a phosphorylation modification, and the first base at the 5' end is A or T. For mouse cell samples, the present invention selected 19,000 genes and some lncRNAs, determined specific targeting sequences through bioinformatics evaluation, and synthesized targeted dual probes.

[0037] Taking the mouse gene Actb as an example, the sequence information of the probe is shown in the following table: 2. Cell Fixation, Perforation, and Probe Hybridization (1) Cell fixation Take 2x10 5 ~2x10 6 Mouse spleen cells were fixed with 4% paraformaldehyde (PFA) at room temperature for 1 hour, centrifuged at 1500 g for 5 minutes at 4°C, and washed three times with PBS for 5 minutes each to remove residual PFA.

[0038] (2) Cell punching Prepare the perforation buffer, which consists of 0.25% (v / v) Triton X-100 (to permeabilize the plasma membrane and nuclear membrane), 50 mM NaCl (to maintain ionic strength and enhance permeability), 250 mM sucrose (to maintain osmotic balance and prevent cell shrinkage), 10 mM HEPES (pH 7.4), 1 mM MgCl2 (to stabilize nuclear structure), and 1 mM CaCl2 (to stabilize membrane tension and regulate nuclear pore function).

[0039] After fixation, cells were incubated in perforation buffer at room temperature for 10 minutes. Washed three times with PBS for 5 minutes each time, and resuspended in hybridization buffer (10% dextran sulfate, 20% formamide, 2× sodium citrate buffer (SSC)).

[0040] (3) Probe hybridization Add the first and second probes to a final concentration of 1 μM each, and an RNase inhibitor to a final concentration of 1 U / μl. Add water to bring the hybridization volume up to 100 μl. Mix thoroughly by pipetting, ensuring that the hybridization solution is free of bubbles. Incubate in a PCR machine at 42°C for 16-24 hours (preheat the PCR machine in advance).

[0041] Preheat wash buffer (10% formamide, 2× sodium citrate buffer (SSC)) in a 42°C metal bath. After hybridization, add wash buffer for washing, incubate in a 42°C metal bath for 10 minutes, and centrifuge at 1500 g for 5 minutes. Wash once more with wash buffer and resuspend in 500 μL of wash buffer.

[0042] (4) Cell counting after hybridization Filter the hybridized cells using a Flowmi® cell strainer and count them using a cell counter to determine the cell concentration and total amount.

[0043] 3. Preparation of Single-cell Water-in-Oil Droplets and Enzymatic Reactions (1) Prepare the enzymatic reaction mix according to the following table: (2) Preparation of cell mix: Prepare a cell mix system based on the amount of cells to be tested (taking 10,000 cells as an example) and the cell concentration, totaling 40 μl.

[0044] (3) Preparation of single-cell oil-in-water droplets Add 70ul of cell phase (enzymatic reaction mix and cell mix), 50ul of beads, and 45ul of Partitioning Oil to the chip, place it in the instrument, and generate water-in-oil droplets.

[0045] (4) Ligation and extension enzymatic reactions Transfer all generated water-in-oil droplets to a PCR tube and perform the enzymatic reaction as follows: 4. Demulsification and Barcoded Probe Pre-amplification (1) After the enzymatic reaction is completed, add 125 μL of recovery agent (1% PFO) to the droplets in the PCR tube and mix thoroughly by inverting to break the emulsion droplets and release the contents of the droplets. The oil and water phases will separate, and the water phase containing the barcoded probe will be located on the upper layer of the oil phase.

[0046] (2) After standing for 2 minutes, centrifuge immediately and discard the 125ul oil phase at the bottom, leaving the aqueous phase for direct reaction.

[0047] (3) Add Amp Mix (purchased from NEB) and pre-amplification primers to the above aqueous phase to perform pre-amplification reaction. The procedure is as follows: (4) Recycling After the reaction, 70 μl of SPRIselect magnetic beads were added to the PCR tube to recover the pre-amplified product, and finally eluted with 50 μl of NFW.

[0048] 4. Library Construction (1) Index PCR amplification Take 20ul of the purified product after pre-amplification, add Amp Mix and index amplification primers to perform amplification reaction. The procedure is as follows: (2) Library fragment screening After index amplification, 100 μl of SPRIselect magnetic beads were added to the PCR tube to recover the amplified product, and finally eluted with 40 μl of NFW.

[0049] 5. Library Quality Control and Sequencing Results Analysis (1) Library quality control The library was quality controlled using an Agilent 2100 Bioanalyzer using microfluidics technology. Single-cell transcriptome sequencing library fragments were distributed within the range of 250–1000 bp, with the main band between 350 and 400 bp and no contamination by small fragments less than 100 bp, indicating a qualified quality control.

[0050] (2) Sequencing results The sequencing data was analyzed using CelCode3Pv1 software, and the analysis results are shown in the following table: Example 2 Best Punch Agent Test The cell pore-forming method was modified to eliminate the pore-forming agent, select saponin (concentration 0.02%) as the pore-forming agent, which pores the plasma membrane much more efficiently than the nuclear membrane, use Triton X-100 at concentrations of 0.1%, 0.3%, and 0.5%, and 70% ethanol as an alternative pore-forming agent. Six mouse genes were selected for experimental testing using mouse PBMCs as samples. The remaining experimental procedures and steps were the same as in Example 1. After the sequencer was removed from the machine, the corresponding UMI median values (100 cells) were analyzed. Additionally, T4 DNA ligase was used instead of SplintR ligase for the experimental testing. The remaining experimental procedures and steps were the same as in Example 1. The results are as follows. The above analysis shows that when only the plasma membrane is perforated, but not the nuclear membrane, the median UMI values of detected genes are significantly reduced, indicating that not all transcriptome RNAs can be amplified in this scenario, preventing the complete transcriptome from being obtained. Furthermore, Triton X-100 is crucial for achieving simultaneous perforation of both the plasma membrane and the nuclear membrane while maintaining overall cell structure (at concentrations above 0.3%, cells begin to lyse, leading to transcriptome loss). When Triton X-100 is replaced with 70% ethanol, another perforating agent that also permeabilizes the nuclear membrane, the median UMI values of detected genes are lower than those obtained with Triton X-100, indicating that Triton X-100 perforation is crucial for obtaining a complete transcriptome. Furthermore, when T4 DNA ligase is used, due to its lack of specificity for RNA-fixed single-stranded DNA, after nuclear membrane perforation, T4 DNA ligase also recognizes and ligates probes hybridized to genomic nucleic acids, resulting in significant nonspecific amplification and a high false-positive rate for transcriptome targeting.

[0051] Example 3 Optimal probe base design test The probes in this invention are single-stranded DNA probes, comprising a first probe and a second probe, with lengths between 25 and 50 nt. While ensuring capture specificity, they offer high stability and reduced cost. To test whether the choice of the first base at the 5'-prime end of the second probe affects detection results, six mouse genes were selected using mouse PBMC as an example. While ensuring consistency between the capture segment and probe length, the first base of the second probe was chosen to be C / G and A / T, respectively. The remaining experimental procedures and steps were the same as in Example 1. After sequencing, the corresponding UMI median values (100 cells) were analyzed using the following analysis results. The analysis results show that genes detected by the second probe with an A or T as the first base at the 5-prime end have a higher median UMI value (per 100 cells), indicating that probes with an A or T as the first base at the 5-prime end have better capture efficiency for target sequences. Therefore, the present invention ultimately prefers designing the second probe with an A or T as the first base at the 5-prime end.

[0052] Example 4 Optimal probe concentration test To test the effect of different probe concentrations on the results, experiments with different probe concentration gradients were conducted to determine the optimal probe concentration. The final concentrations of the first and second probes were set to 0.1 μM, 0.5 μM, 1 μM, 1.5 μM, and 2 μM, respectively. Other variables and experimental procedures were the same as in Example 1. The results obtained by CelCode3Pv1 software analysis are as follows. The analysis results show that the number of genes detected and the median UMI are low at low probe concentrations. Higher probe concentrations detect more effective information, but at a certain probe concentration, there is no significant increase in the number of genes detected. Considering probe redundancy and the high cost of synthesis, the optimal probe concentration for this invention is between 0.8 and 1.2 μM, with a final probe concentration of 1 μM being selected.

[0053] Example 5 Sensitivity Test To test the sensitivity of this method for RNA detection in samples, SW620 cells were selected for library construction. A cell gradient of 1800 cells, 900 cells, 600 cells, and 300 cells was set up. Hybridization capture and library construction were performed as in Example 1, followed by relative quantification using fluorescent quantitative PCR.

[0054] The results are as follows Figure 4 As shown, the linear correlation R2 of the gene expression Ct values of the cell samples is 0.9929, which proves that the method of the present invention can effectively detect RNA expression in a small amount of cells and has a good linear relationship.

[0055] Example 6 To verify the differences in gene detection between this method and conventional single-cell three-step transcriptome sequencing, a comparative experiment was conducted using mouse brain cells. Group 1 followed the conventional single-cell three-step transcriptome sequencing protocol, while Group 2 followed the protocol of Example 1. The estimated number of cells in both groups was 10,000. Data analysis was performed after the analysis, and the comparative results are shown in the table below. The analysis results show that due to the limitations of the targeted capture experimental design, the total number of genes detected by this method is not as good as that of conventional methods. However, in terms of the gene median and UMI median, which are of greater concern to researchers, the results of this method are better than those of existing conventional detection methods.

[0056] It should be noted that the preferred embodiments of the present invention are given in the specification and drawings of the present invention. However, the present invention can be implemented in many different forms and is not limited to the embodiments described in this specification. These embodiments are not intended to be additional limitations on the content of the present invention. The purpose of providing these embodiments is to make the understanding of the disclosure of the present invention more thorough and comprehensive. In addition, the above-mentioned technical features can be combined with each other to form various embodiments not listed above, which are all considered to be within the scope of the description of the present invention. Furthermore, it is obvious to those skilled in the art that improvements or changes can be made based on the above description, and all such improvements and changes should fall within the scope of protection of the claims attached to the present invention.

Claims

1. A method for constructing a high-throughput single-cell transcriptome library based on probe-targeted enrichment, characterized in that: The following steps are involved: (1) Providing one or more cells or cell nuclei, fixing them and performing perforation treatment; The pore punching treatment is performed using a Triton X-100 solution having a volume concentration between 0.1% and 0.3%; (2) contacting the fixed cells or cell nuclei with one or more nucleic acid-specific targeting dual probes, wherein the nucleic acid-specific targeting dual probes comprise a first probe and a second probe, wherein the first probe comprises a 3'-end nucleic acid-specific targeting sequence and a first universal sequence, and the second probe comprises a 5'-end nucleic acid-specific targeting sequence and a first linker sequence, so that the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridize with a first target region and a second target region of a target RNA transcript in the cell or cell nucleus, respectively, and the first target region is adjacent to the second target region; (3) mixing the cells or cell nuclei treated in step (2) with the enzymatic reaction mix to obtain a cell phase, which is then mixed with beads and an oil phase to prepare water-in-oil droplets; The enzymatic reaction mix comprises a ligation reagent that allows the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridized with the target RNA transcript to be ligated together to form an RNA-probe double strand, a reagent that removes RNA from the RNA-probe double strand, and an extension reagent; The beads are connected with a barcode sequence, a second universal sequence and a second connection sequence; (4) performing an enzymatic reaction in the water-in-oil droplet, so that the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridized with the target RNA transcript are linked together to form an RNA-probe double strand, RNA is removed from the RNA-probe double strand, and the first linker sequence is hybridized with the second linker sequence and extended along the probe chain to obtain a barcoded probe; (5) Demulsification is performed to recover the barcoded probes, pre-amplification is performed, and the enriched fragments are labeled using Index PCR. After product purification, a single-cell transcriptome library that can be directly used for sequencing is obtained.

2. The method according to claim 1, characterized in that The fixation treatment is performed using paraformaldehyde at a concentration of 0.5%-8%.

3. The method according to claim 1, characterized in that The first base at the 5' end of the second probe is A or T; and the 5' end of the second probe contains phosphorylation modification.

4. The method according to claim 1, wherein The target RNA transcripts include mRNA and non-coding RNA.

5. The method according to claim 1, wherein The ligation reagent that ligates the 3'-end nucleic acid-specific targeting sequence and the 5'-end nucleic acid-specific targeting sequence hybridized with the target RNA transcript to form an RNA-probe double strand is SplintR ligase.

6. The method according to claim 1, characterized in that The reagent for removing RNA from the RNA-probe double strand includes RNase H.

7. The method according to claim 1, characterized in that The extension reagent includes dNTPs, DNA polymerase or Mu polymerase.

8. The method according to claim 1, characterized in that The pre-amplification is performed using pre-amplification primers targeting the first universal sequence and the second universal sequence.

9. A high-throughput single-cell transcriptome library constructed by the method according to any one of claims 1 to 8.

10. Use of the high-throughput single-cell transcriptome library according to claim 9 in single-cell sequencing.

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