Single cell time transcriptome sequencing method based on RNA double metabolism markers

Abstract

The invention discloses a single-cell time transcriptome sequencing method based on RNA (Ribonucleic Acid) bimetabolism markers. The single-cell time transcriptome sequencing method comprises the following steps: (1) obtaining single cells of 6sG and 4sU bimetabolism markers; (2) loading the marked and fixed cells and the coding microspheres onto a double-layer micropore array chip, cracking the cells, and capturing dissociated RNA (Ribonucleic Acid) by the coding microspheres; (3), carrying out TimeLapse chemistry processing on the coding microspheres, and carrying out the TimeLapse chemistry processing on the coding microspheres; (4) sequentially carrying out reverse transcription, cDNA amplification, library construction and sequencing on the material obtained in the step (3), and then carrying out bioinformatics analysis to identify the mutation marker; and (5) for the state or the cell type of each cell, calculating the RNA degradation rate according to a curve that the proportion of reads carrying G-to-A mutation decreases, and deriving the RNA synthesis rate according to a curve that the proportion of reads carrying T-to-C mutation increases. By combining a 6sG and 4sU double-pulse labeling technology and high-throughput single cell sequencing, synchronous measurement of RNA synthesis and degradation dynamics on the single cell level is realized.

Description

Technical Field

[0001] The present invention belongs to the technical fields of gene sequencing and molecular biology, and particularly relates to a single-cell time transcriptome sequencing method based on RNA dual metabolic labeling. Background Art

[0002] Gene expression changes with cell differentiation, development process, and external stimuli. During this process, mammalian cells regulate the synthesis and degradation rates of RNA through multiple strategies, so as to achieve fine regulation of RNA levels, and further achieve dynamic gene expression. Higher RNA synthesis or degradation rates enable cells to quickly respond to various signals from inside and outside the cell, which is crucial for the rapid adaptation and functional regulation of cells. While lower RNA degradation rates allow cells to accumulate more transcriptional information, which is of great significance for maintaining cell stability and long-term function. Starting from the information in the time dimension can help this embodiment understand more deeply how cells achieve precise gene expression through the dynamic regulation of RNA synthesis and degradation processes. Therefore, conducting time transcriptome sequencing research including RNA synthesis and degradation processes has important scientific significance and application value.

[0003] Single-cell transcriptome sequencing technology has important application value in studying gene expression at the single-cell level and revealing cell heterogeneity. It can accurately distinguish different cell subtypes, infer cell differentiation trajectories, and deeply explore gene expression and regulation mechanisms. This makes single-cell transcriptome sequencing show broad application prospects in multiple fields such as developmental biology, immunology, neuroscience, and clinical medicine. For example, in developmental biology, it can reveal the formation and functional differentiation of different cell types during embryonic development; in immunology, it can analyze the activation and differentiation mechanisms of immune cells; in neuroscience, it can study the gene expression differences between neurons and glial cells and their effects on the function of the nervous system; in clinical medicine, it can assist in disease diagnosis, treatment, and prognosis assessment. However, most of the existing single-cell transcriptome analysis methods can only capture the instantaneous gene expression state of cells and cannot obtain continuous gene expression dynamic information in the time dimension, which limits people's comprehensive understanding of the cell development and differentiation process.

[0004] The dynamic analysis technology of transcriptome based on RNA metabolic labeling can capture the dynamic changes of gene expression in real time, becoming an ideal tool for studying the single-cell temporal transcriptome. This method labels nascent RNA with unnatural nucleosides, and through subsequent chemical treatment and high-throughput sequencing, obtains the expression information of nascent RNA, thereby realizing the measurement of the synthesis and degradation rates of RNA. However, most of the existing research methods rely on a single labeling strategy and cannot measure the synthesis and degradation rates of RNA simultaneously, which limits the comprehensive understanding of the RNA dynamic regulation mechanism. In addition, the single labeling strategy may lead to an overestimation of the RNA half-life, thus affecting the accurate assessment of the dynamic changes in gene expression.

[0005] In a paper published in *Angewandte Chemie* by Alexandra Lusser et al., a dual metabolic labeling method (TUC-seq DUAL) was proposed for evaluating mRNA lifespan. This method combines the dual metabolic labeling of 6sG and 4sU and OsO 4 -hydrazine chemical treatment to achieve high-resolution detection of mRNA dynamic changes. However, there are the following problems with this technical solution: First, the G-to-A mutation conversion rate is relatively low, and the proportion of transcripts carrying G-to-A mutations does not show a time-dependent decrease, making it difficult to meet the requirements of simultaneously measuring the synthesis and degradation rates of RNA at the single-cell level. Second, the chemical treatment process is complex, requiring a large variety of reagents and a cumbersome preparation process, and the overall treatment process takes a long time, which may lead to RNA degradation, significantly reducing the detection efficiency of trace RNA, and thus affecting the accuracy and reproducibility of the data. In addition, multiple transfers, centrifugations, and reactions are required in the specific operation, which has a high operation risk in single-cell processing and may introduce additional errors and contamination. Currently, there is no technical solution that can achieve efficient RNA double mutations at single-cell resolution to simultaneously measure the synthesis and degradation rates of RNA. Summary of the Invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide a single-cell temporal transcriptome sequencing method based on RNA dual metabolic labeling.

[0007] The technical solution of the present invention is as follows:

[0008] A single-cell temporal transcriptome sequencing method based on RNA dual metabolic labeling, comprising the following steps:

[0009] (1) Incubate and label cells with 6-thioguanosine and 4-thiouridine successively to obtain single cells with dual metabolic labeling;

[0010] (2) Load the above single cells that have been labeled and fixed with methanol together with the encoded microspheres onto a double-layered microwell array chip, lyse the cells using a solid-state lysis solution, and the double-metabolic-labeled RNA released is captured by the above encoded microspheres;

[0011] (3) Mix the encoded microspheres obtained from step (2), 5×wash buffer solution, TFEA solution, NaIO 4 solution and DEPC-treated water to form a reaction system of 25 - 30 μL, and incubate the reaction to perform TimeLapse chemistry treatment;

[0012] (4) After subjecting the materials obtained in step (3) to reverse transcription, cDNA amplification, library construction, and sequencing in sequence, perform bioinformatics analysis to identify mutation markers;

[0013] (5) For each cell state or type, use the curve of the decreasing proportion of reads carrying G-to-A mutations to calculate the corresponding RNA degradation rate, and use the curve of the increasing proportion of reads carrying T-to-C mutations to deduce the corresponding RNA synthesis rate.

[0014] In a preferred embodiment of the present invention, step (1) includes: incubating and labeling the cells to be tested with a medium containing 0.15 - 0.25 mM 6-thioguanosine for 10 - 12 h, and then incubating and labeling with a medium containing 0.15 - 0.25 mM 4-thiouridine for 0.5 - 2 h.

[0015] In a preferred embodiment of the present invention, the preparation of the 5×wash buffer solution is as follows: dissolve sodium acetate solution and ethylenediaminetetraacetic acid solution in DEPC-treated water, wherein: the concentration of the sodium acetate solution is 2.8 - 3.2 M, and the pH is 5.0 - 5.4; the concentration of the ethylenediaminetetraacetic acid solution is 0.4 - 0.6 M, and the pH is 7.5 - 8.4.

[0016] More preferably, the volume ratio of the sodium acetate solution, ethylenediaminetetraacetic acid solution, and DEPC-treated water is 7 - 8:1 - 3:120 - 130.

[0017] Even more preferably, the concentration of the TFEA solution is 5.5 - 6.5 M, and the concentration of the NaIO 4 solution is 95 - 105 mM.

[0018] Even further preferably, in step (2), the volume ratio of the 5×wash buffer solution, TFEA solution, NaIO 4 solution and DEPC-treated water is 4 - 6:2 - 3:2 - 3:13 - 17.

[0019] In a preferred embodiment of the present invention, the temperature of the incubation reaction in step (2) is 42 - 47 °C, and the time is 0.8 - 1.2 h.

[0020] In a preferred embodiment of the present invention, the preparation of the 5×wash buffer solution is as follows: Dissolve the sodium acetate solution and the ethylenediaminetetraacetic acid solution in DEPC-treated water, wherein: the concentration of the sodium acetate solution is 3 M, and the pH is 5.2; the concentration of the ethylenediaminetetraacetic acid solution is 0.5 M, and the pH is 8.0; the volume ratio of the sodium acetate solution, the ethylenediaminetetraacetic acid solution, and the DEPC-treated water is 8:2:127.

[0021] More preferably, the concentration of the TFEA solution is 6 M, and the concentration of the NaIO 4 solution is 100 mM.

[0022] The beneficial effects of the present invention are as follows:

[0023] 1. By optimizing the 4sU labeling and chemical treatment conditions, the present invention significantly improves the efficiency of G-to-A and T-to-C mutations. The experimental results show that the highest proportion of transcripts carrying G-to-A mutation markers and transcripts carrying T-to-C mutation markers detected in a single gene can reach about 25% respectively. This changing trend conforms to the theoretical expectation and can be used to accurately calculate the synthesis and degradation rates of RNA.

[0024] 2. The chemical treatment time of the present invention is only 1 h, and the reagent preparation is simple, requiring few instruments and low cost. Although the 6sG labeling time is longer, the total of the labeling time and the chemical treatment time only needs 15 h, which is significantly less than the treatment time required by the existing methods, greatly improving the experimental efficiency.

[0025] 3. By combining the 6sG and 4sU dual-pulse labeling techniques with high-throughput single-cell sequencing, the present invention realizes the synchronous measurement of RNA synthesis and degradation dynamics at the single-cell level. This can reveal specific RNA regulation strategies in different cell types or cell states, providing a new perspective for studying the heterogeneity in complex biological systems, the functions of rare cell populations, and transcriptional regulation during cell state transitions. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 Show the 4sU and 6sG structures in Example 1 of the present invention and their corresponding product structures after being treated with TimeLapse chemistry.

[0027] Figure 2Show in Example 1 of the present invention: a. 1H NMR spectra of 4TU before (upper figure) and after (lower figure) TimeLapse chemistry treatment; b. 1 1H NMR spectra of 6TG before (upper figure) and after (lower figure)

[0028] Figure 3 Show the workflow of scDUAL-seq in Example 2 of the present invention.

[0029] Figure 4 Show the mutation rate distribution at different labeling time points in Example 2 of the present invention.

[0030] Figure 5 Show the clustering heatmap of RNA synthesis rate, degradation rate and total RNA level of 9,325 genes in the cell cycle process in Example 2 of the present invention; the left band shows the cosine similarity between the RNA synthesis rate and the degradation rate. Detailed implementation manners

[0031] The technical solutions of the present invention will be further described and illustrated below through specific implementation manners in combination with the accompanying drawings.

[0032] Example 1

[0033] TimeLapse chemistry treatment:

[0034] Simon et al. developed TimeLapse chemistry. In this method, the thiol group of 4sU is oxidized by sodium periodate (NaIO 4 ) and reacts with 2,2,2-Trifluoroethanol (TFEA) to generate trifluoroethylated cytidine, realizing the T-to-C mutation on the 4sU-labeled transcript. They further confirmed that TimeLapse chemistry is also applicable to the conversion of 6-thioguanine (6TG), thus generating G-to-A mutations in 6TG-labeled RNA. Since 4sU and 6sG have similar thiol structures and 6sG binds more easily to nascent RNA than its base 6TG, this example attempts to apply TimeLapse chemistry to the chemical conversions of 4sU and 6sG simultaneously (as Figure 1 shown).

[0035] Operation method: Sodium acetate buffer (CH 3COONa buffer; 8 μL, 3 M, pH 5.2) and ethylenediaminetetraacetic acid buffer (EDTA buffer; 2 μL, 0.5 M, pH 8.0) were dissolved in 127 μL of DEPC-treated water to prepare 5× wash buffer. After preparation, it could be placed in a centrifuge tube and stored at 4 °C for multiple uses. Freshly prepared sodium periodate buffer (NaIO 4 buffer; 100 mM) and 2,2,2-Trifluoroethanol buffer (TFEA buffer; 6 M). The coding microspheres (10,000 - 30,000, from single-cell experiments) carrying double-metabolically labeled RNA were mixed with 5 μL of 5× wash buffer solution, 2.5 μL of TFEA solution, 2.5 μL of NaIO 4 solution and 15 μL of DEPC-treated water to obtain a mixture with a total volume of 25 μL. The mixture was incubated at 45 °C for 1 h to complete the chemical treatment step.

[0036] To preliminarily verify the possibility of TimeLapse chemistry to achieve chemical nucleoside conversion, in this example, 4-thiouracil (4TU; 4.3 mg, 1 equiv) was dissolved in DMSO-d 6 . Subsequently, TFEA (3.4 μL, 1.3 equiv) was added. After mixing, NaIO 6 dissolved in DMSO-d 4 (12.3 mg, 1.7 equiv) was added. The reaction was carried out at 45 °C for 1 h. Similarly, 6TG (10.7 mg, 1 equiv) was dissolved in DMSO-d 6 . TFEA (6.38 μL, 1.2 equiv) was added. After mixing, NaIO 6 dissolved in DMSO-d 4 (20.5 mg, 1.5 equiv) was added. The reaction was carried out at 45 °C for 1 h. Finally, the obtained 1 1H nuclear magnetic resonance (NMR) spectra were analyzed using MestReNova software. As Figure 2As shown in a, the NMR spectrum of 4TU after TimeLapse chemistry treatment showed an upregulation of two proton peaks, and there were signal peaks belonging to TFEA. Similar results were also observed in the TimeLapse chemistry conversion of 6TG (as shown in Figure 2 b). These results confirmed the ability of TimeLapse chemistry to convert 4TU and 6TG into cytosine and adenine analogs.

[0037] Example 2 Application at the single-cell level (scDUAL-seq)

[0038] (1) Dual metabolic labeling: 6-Thioguanosine (6sG) and 4-thiouridine (4sU) are synthetic compounds with chemical structures highly similar to natural ribonucleosides (guanosine (G) and uridine (U)). During RNA synthesis, 6sG and 4sU can respectively base pair with cytidine (C) and adenosine (A) through hydrogen bonding and be recognized by RNA polymerase, and be incorporated into the nascent RNA strand as alternative bases of the template strand.

[0039] Operation method: HCT116 cells (human colorectal cancer cells (ATCC, CCL-247)) were labeled with a medium containing 0.2 mM 6sG at 37 °C for 12 h (the first pulse), and then the medium was replaced with a medium containing 0.2 mM 4sU to incubate the cells (the second pulse). Cells were collected after 12 h of 6sG incubation, 0.5 h, 1 h, and 2 h of 4sU incubation to correspond to different experimental groups (as shown in Figure 3 a).

[0040] (2) Cell fixation and treatment. Resuspend 1×10 6 to 5×10 6 doubly metabolically labeled cells in 100 μL of ice-cold 1×PBS (containing 40 U RNase Inhibitor), then add 900 μL of ice-cold methanol, and fix in the dark on ice for 10 min. After fixation, the cells were centrifuged at 900 g at 4 °C for 3 min to collect the precipitate. The cell precipitate was washed once with ice-cold 1×PBS and resuspended in 1×PBS containing 0.05% bovine serum albumin, and the concentration was adjusted to 200 cells / μL.

[0041] (3) Library construction and sequencing: Load the labeled and fixed cells obtained in step (2) onto a double-layer microwell array chip (Decoder Cartridge; Dynamic Biosystems, 2203106)(Figure 3 a), and library preparation was carried out using the DECODER single-cell 3' end kit (Dynamic Biosystems, 2203206, 2203306, 2203406). The TimeLapse chemistry processing step in Example 1 was additionally added before reverse transcription ( Figure 3 b), and the rest was completed in accordance with the kit instructions.

[0042] Briefly, individual cells and individual encoded microspheres were automatically loaded into a double-layered microwell array chip in sequence by the DECODER single-cell system (Dynamic Biosystems, 13030003-A1). A solid-state lysis solution was further automatically introduced into the chip to lyse the cells, and the released RNA was immediately captured by the microspheres. The microspheres were recovered from the chip and washed successively with 6× saline sodium citrate (SSC) and 1× washing buffer. The washed microspheres were then subjected to TimeLapse chemistry conversion as described in Example 1. Then the microspheres were washed once with TE buffer (10 mM Tris-HCl, 1 mM EDTA) and incubated in 50 μL of a reducing solution (100 mM NaCl, 10 mM DTT, and 1.6 U / μL of RNase inhibitor added to TE buffer) at 30 °C for 30 min, and then washed three times with 200 μL of 6× SSC and once with 100 μL of RTreagent A reagent (Dynamic Biosystems, 2203306).

[0043] After TimeLapse chemistry treatment, the microspheres were subjected to reverse transcription, cDNA amplification, and library construction using the reagents in the DECODER single-cell 3' end kit (Dynamic Biosystems, 2203306, 2203406) without additional modification. The quality of the library was detected by Qsep-100, and then paired-end sequencing (150 bp) was performed on the Illumina NovaSeq 6000 platform.

[0044] (4) Identifying mutation markers: Analyze the sequencing results using bioinformatics analysis, and label the cell barcode carried by read1 and the Unique Molecular Identifier (UMI) to the mRNA reads of read2. Remove the sequencing adapters and poly(A) sequences, and then use STAR to align the sequencing reads with the human reference genome GRCh38 to identify the mutation sites that are different from the reference genome sequence, thereby identifying the reads with different mutation markers (such as Figure 4 as shown).

[0045] (5) For each gene, calculate its GA reads count, TC reads count, and GA&TC reads count, and pool them into a matrix, where the rows of the matrix are gene names and the columns are cell barcodes. Divide the GA reads count by the total UMI count to calculate the GA reads proportion of each gene. Use the nls function in R for fitting, where the fitting formula is set to y~I(a×exp(-b×x)), the initial parameters are set to start=list(a=1, b=0), and the missing value parameter is set to na.action=na.exclude. Only retain the genes with a goodness of fit R 2 >0.4 for further analysis.

[0046] The degradation rate constant (γ, unit: / h) is derived using the following formula through the RNA half-life (t 1 / 2 ):

[0047]

[0048] Then, assuming that the gene-specific RNA synthesis rate (α, unit: molecules / h) remains constant for all cells within a given cell state, calculate the synthesis rate according to the following formula:

[0049]

[0050] where n represents the average value of the sum of the TC reads count and the GA&TC reads count of the gene in a specific cell cycle or cell state, γ is the gene-specific degradation rate constant in a specific cell cycle or cell state, and t (unit: h) is the metabolic labeling time. In this way, the calculation of the RNA synthesis rate and the degradation rate is achieved (such as Figure 5 shown).

[0051] As described above, it is only the preferred embodiment of the present invention, so the scope of implementation of the present invention cannot be limited thereby. That is, equivalent changes and modifications made according to the scope of the present invention patent and the content of the specification should still fall within the scope covered by the present invention.

Claims

1. A single-cell temporal transcriptome sequencing method based on RNA dual metabolic labeling, characterized in that: The steps include: (1) The cells are incubated and labeled with 6-thioguanosine and 4-thiouridine successively to obtain dual metabolically labeled single cells; (2) loading the labeled and methanol-fixed single cells together with the encoded microspheres onto a double-layer microwell array chip, lysing the cells with a solid lysis solution, and capturing the dissociated dual-metabolism labeled RNA with the encoded microspheres; (3) Mix the encoded microspheres obtained in step (2), 5× wash buffer solution, TFEA solution, NaIO4 solution and DEPC-treated water to form a 25-30 μL reaction system, incubate the reaction, and perform TimeLapse chemistry treatment; (4) performing reverse transcription, cDNA amplification, library construction and sequencing on the material obtained in step (3), and then performing bioinformatics analysis to identify mutation markers; (5) For each cell state or type, the curve with a decreasing proportion of reads carrying G-to-A mutations is used to calculate the corresponding RNA degradation rate, and the curve with an increasing proportion of reads carrying T-to-C mutations is used to deduce the corresponding RNA synthesis rate.

2. The single-cell temporal transcriptome sequencing method according to claim 1, characterized in that: The step (1) comprises: firstly incubating the cells to be tested with a culture medium containing 0.15-0.25 mM 6-thioguanosine for 10-12 hours, and then incubating with a culture medium containing 0.15-0.25 mM 4-thiouridine for 0.5-2 hours.

3. The single-cell temporal transcriptome sequencing method according to claim 1, characterized in that: The 5×wash buffer solution is prepared by dissolving a sodium acetate solution and an ethylenediaminetetraacetic acid solution in DEPC-treated water, wherein the concentration of the sodium acetate solution is 2.8-3.2 M, and the pH is 5.0-5.4; the concentration of the ethylenediaminetetraacetic acid solution is 0.4-0.6 M, and the pH is 7.5-8.

4.

4. The single-cell temporal transcriptome sequencing method according to claim 3, characterized in that: The volume ratio of sodium acetate solution, ethylenediaminetetraacetic acid solution and DEPC treated water is 7-8:1-3:120-130.

5. The single-cell temporal transcriptome sequencing method according to claim 4, characterized in that: The concentration of the TFEA solution is 5.5-6.5 M, and the concentration of the NaIO4 solution is 95-105 mM.

6. The single-cell temporal transcriptome sequencing method according to claim 5, characterized in that: In the step (3), the volume ratio of 5×wash buffer solution, TFEA solution, NaIO4 solution and DEPC treated water is 4-6:2-3:2-3:13-17.

7. The single-cell temporal transcriptome sequencing method according to any one of claims 1 to 6, characterized in that: The incubation reaction temperature of step (3) is 42-47° C. and the incubation time is 0.8-1.2 h.

Images