Low-abundance cell nucleus enrichment method

Abstract

The invention discloses a low-abundance cell nucleus enrichment method which comprises the following steps: (1) splitting a tissue sample to obtain a cell nucleus with extracellular cytoplasm retained; and (2) marking and sorting the cell nucleuses obtained in the step (1) to obtain enriched low-abundance cell nucleuses. According to the method, a mild dissociation technology is used, so that more cytoplasm components can be reserved in cell nucleuses, and then the cell nucleuses are used for marking cytoplasm proteins and are combined with flow sorting, so that directional enrichment of the cell nucleuses with low abundance and specific functional states can be effectively realized. Through the whole process optimization, the method can ensure the completeness of the cell nucleus to the maximum extent, takes downstream sequencing application into consideration, and can obtain high-quality mononuclear transcriptome sequencing data by combining with the screening of cytoplasm sequencing data. Moreover, according to the method, directional enrichment is realized through the cytoplasmic protein, the use cost can be effectively reduced, and the method is suitable for large-scale clinical sample data acquisition and has higher flux compared with a single space transcriptome slice.

Description

Technical Field

[0001] This invention relates to the field of biomedicine, and in particular to a method for enriching low-abundance cell nuclei. Background Technology

[0002] Single-cell and single-nuclear transcriptome sequencing (sc / snRNA-seq) has become a key technology for elucidating tissue cellular heterogeneity. Among them, single-nuclear sequencing is particularly important in brain science research due to its good compatibility with samples such as frozen tissues. However, highly specific enrichment of low-abundance nuclear populations within tissues remains a core bottleneck in this field. Existing methods mainly rely on nuclear chromatin or DNA dyes for sorting, which makes it difficult to distinguish nuclei from different cell sources, and the processing may damage RNA integrity, resulting in poor enrichment efficiency and reproducibility. This problem is particularly prominent for research on diseases such as Parkinson's disease: key pathological neurons labeled with phosphorylated α-synuclein (p-α-Syn) in the substantia nigra pars compacta (SNc) of the midbrain are extremely rare, and this label is mainly found in the cytoplasm. Traditional nuclear feature-based sorting or spatial transcriptome techniques are insufficient for efficient and specific enrichment and sequencing of cell nuclei.

[0003] To overcome this limitation, emerging technologies have begun to explore enrichment strategies that do not rely on cell surface or cytoplasmic proteins, such as the PERFF-seq method. This method utilizes flow cytometry in situ hybridization (FlowFISH) technology targeting specific RNA transcripts to programmatically enrich nuclear populations defined by low-abundance RNA markers directly from cell nuclei isolated from cryopreserved or FFPE tissues, and is compatible with downstream high-throughput single-nucleus transcriptome sequencing. However, this method suffers from problems such as operational complexity, low throughput, and high cost, and it is difficult to accurately correspond to specific cell types and to efficiently enrich rare cell nuclei. Summary of the Invention

[0004] Purpose of the invention: The purpose of this invention is to provide a low-cost, high-throughput, and easy-to-operate method for effectively achieving high-quality enrichment of low-abundance cell nuclei.

[0005] Technical solution: The method for enriching low-abundance cell nuclei in a sample according to the present invention includes the following steps: (1) Lyse the tissue sample to obtain cell nuclei with the extranuclear cytoplasm retained; (2) The cell nuclei obtained in step 1 are labeled and sorted to obtain enriched low-abundance cell nuclei.

[0006] Preferably, step 1 includes: (11) The tissue sample was mixed with the lysis buffer and homogenized to obtain the lysis product; (12) Filter the lysate obtained in step 11, centrifuge and resuspend to obtain a cell nucleus suspension that retains the extranuclear cytoplasm.

[0007] Preferably, the lysis buffer in step 11 contains 145-147 mM NaCl, 20-22 mM MgCl2, 9-11 mM Tris-HCl, 0.5-1.5 mM CaCl2, 0.008-0.012% w / v bovine serum albumin, 0.025-0.035% v / v polysorbate-20, a pharmacologically effective concentration of a protease inhibitor, a pharmacologically effective concentration of an RNase inhibitor, and the remainder is water; the homogenization step is performed using the type A pestle of a Dounce homogenizer; more preferably, the RNase inhibitor is any one of diethyl pyrocarbonate, guanidine isothiocyanate, vanadate ribonucleoside complex, and a protein inhibitor of RNase; the gap between the type A pestle and the cylinder of the Dounce homogenizer is 0.001-0.003 inches.

[0008] Preferably, the filtration step in step 12 uses a sieve with a pore size of 35~45 μm; the centrifugation conditions are 2~8℃, 500×g centrifugation for 4~6 min.

[0009] Preferably, step 2 includes: (21) Fix the cell nuclei obtained in step 1 with the extranuclear cytoplasm retained, and wash them to obtain fixed cell nuclei; (22) Seal and fix the cell nucleus and label it; (23) The labeled cell nuclei obtained in step 22 are sorted to obtain enriched low-abundance cell nuclei.

[0010] Preferably, the fixation step in step 21 uses methanol for fixation; the washing step is to centrifuge at 2~8℃, 500×g for 4~6 min, discard the supernatant, resuspend in phosphate buffer, centrifuge again at 2~8℃, 500×g for 4~6 min, and discard the supernatant.

[0011] Preferably, the blocking step in step 22 is to block at 2~8℃ for 10~20 min; the labeling step uses fluorescent antibodies and / or fluorescent probes for labeling; more preferably, the fluorescent antibody is a fluorescent antibody against cytoplasmic proteins.

[0012] Preferably, the sorting step in step 23 is performed using flow cytometry.

[0013] The cell nucleus described in this invention is prepared by the aforementioned method.

[0014] The application of the cell nucleus described in this invention in single-nucleus transcriptome sequencing.

[0015] Preferably, the application steps include: constructing an RNA library using the aforementioned cell nucleus and then performing single-nucleus transcriptome sequencing; more preferably, the single-nucleus transcriptome sequencing further includes screening to exclude environmental RNA data contamination.

[0016] Beneficial Effects: Compared with existing technologies, the present invention has the following significant advantages: 1. The method described in the present invention uses a mild dissociation technique, which allows the cell nucleus to retain more cytoplasmic components, which can then be used for cytoplasmic protein labeling. Combined with flow cytometry sorting, it can effectively achieve targeted enrichment of low-abundance, functionally specific cell nuclei; 2. Through overall process optimization, this method can maximize the integrity of the cell nucleus, while also considering downstream sequencing applications. Combined with the screening of cytoplasmic sequencing data, high-quality single-nucleus transcriptome sequencing data can be obtained; 3. This method achieves targeted enrichment through cytoplasmic proteins, which can effectively reduce the cost of use, is suitable for large-scale clinical sample data acquisition, and has a higher throughput compared to a single spatial transcriptome slice. Attached Figure Description

[0017] Figure 1 A schematic diagram of the process for enriching low-abundance cell nuclei; Figure 2 The images show the results of cell nucleus sorting. In the images, A is a representative image of the MAP2 group acquired by confocal fluorescence microscopy, B is a representative image of the DAPI group acquired by confocal fluorescence microscopy, and C is the quantitative results of the MAP2 positive ratio between the MAP2 group and the DAPI group. Figure 3 This is a diagram showing the results of the nuclear sequencing data obtained from the enrichment in Example 1; Figure 4 This is a comparison of the nuclear sequencing data of the MAP2 group and the DAPI group obtained from the enrichment in Example 1. In this figure, A is a cell composition analysis diagram of the DAPI group and the MAP2 group; B is a violin plot comparing the MAP2 expression levels of each cell type in the DAPI group and the MAP2 group; C is a bar chart of the MAP2 expression ratio of each cell type in the DAPI group and the MAP2 group. ns indicates p>0.05, * indicates p≤0.05, ** indicates p≤0.01, *** indicates p≤0.001; **** indicates p≤0.0001. Figure 5 Box plot of mitochondrial gene percentage in the nuclear sequencing data obtained from Example 1 enrichment; Figure 6 This is a graph showing the results of gene set enrichment analysis after sequencing in Example 2; Figure 7 The diagram shows a comparison of the distribution of full-length cDNA amplified by Example 1 and Comparative Example 1. In Example 1, A shows the distribution of full-length cDNA fragments amplified by the method of Example 1, and B shows the distribution of full-length cDNA fragments amplified by the method of Comparative Example 1. Figure 8 The diagram shows a comparison of flow cytometry signals between Example 1 and Comparative Example 2. In Example 1, A is a flow cytometry signal analysis diagram of the cell nucleus obtained by fluorescence staining, and B is a flow cytometry signal analysis diagram of the cell nucleus obtained by fluorescence staining. Detailed Implementation

[0018] The technical solution of the present invention will be further described below.

[0019] Example 1: Nuclear enrichment and single-nuclear transcriptome sequencing of cells with high expression of microtubule-associated protein 2 (MAP2) in mouse brain tissue 1. Nucleus enrichment of cells with high expression of microtubule-associated protein 2 (MAP2) in mouse brain tissue S1, 8 mg of frozen fresh brain tissue from C57BL / 6J mice and 4 mL of lysis buffer were added together into a Dounce glass homogenizer pre-chilled on ice, and lysis was performed by grinding vertically and uniformly with an A pestle 15 times. The lysis buffer was a TSTD solution containing 146 mM NaCl, 21 mM MgCl2, 10 mM Tris-HCl, 1 mM CaCl2, 0.01% w / v bovine serum albumin, 0.03% v / v polysorbate-20 (Tween-20), 0.5% v / v protease inhibitor compound (Beyotime, catalog number P1005), 1% v / v diethyl pyrocarbonate (DEPC), with the remainder being water. S2, a 40 μm sieve was rinsed with 200 μL phosphate-buffered saline (PBS), and then the sieve was used to filter the brain tissue cell nucleus suspension after S1 lysis, and the sieve was rinsed again with 2 mL PBS; The brain cell nucleus suspension filtered from S3 and S2 was centrifuged at 4°C, 500 × g, with an ascending rate of 3 (1470 rpm / min) and a descending rate of 3 (640 rpm / min) for 5 min. The supernatant was discarded, and the system was resuspended with PBS to bring the volume to 200 μL. S4: The brain tissue cell nuclei resuspended in pure methanol pre-cooled at -20℃ for 30 min were fixed in S3. The solution was added dropwise while stirring, and the operation was repeated 4 times, for a total of 800 μL. S5, the cell nucleus suspension containing methanol from S4 was centrifuged at 4℃, 500 ×g, with an ascent rate of 2 (1100 rpm / min) and a deceleration rate of 2 (530 rpm / min) for 5 min. The supernatant was discarded, and the cell nucleus pellet was resuspended in 1.3 mL of PBS. Then, it was centrifuged again at 4℃, 500 ×g, with an ascent rate of 2 (1100 rpm / min) and a deceleration rate of 2 (530 rpm / min) for 5 min. The supernatant was discarded. S6. Add 100 μL of blocking buffer to the nuclear pellet washed in S5, resuspend, and block at 4°C for 15 min. The blocking solution contains PBS, 10% v / v donkey serum for blocking (Solarbio, catalog number #SL050), 2U / μL LNase Inhibitor (New First Production, catalog number LS-EZ-E-00006P), and the remainder is water. S7. The cell nucleus samples blocked in S6 were processed at a concentration of 0.4 μg / 10⁻⁶. 6 Add MAP2 direct-labeled antibody (Proteintech, catalog number CL594-17490) to each cell nucleus, mix well, and incubate at 4°C in the dark for 30 min. Then add 500 μL of PBS, centrifuge at 500 ×g at 4°C, with an ascent rate of 2 (1100 rpm / min) and a deceleration rate of 2 (530 rpm / min) for 5 min, discard the supernatant, add 100 μL of DAPI immunofluorescence buffer, and incubate on ice in the dark for 3 min. The DAPI immunofluorescence buffer contains PBS, 10% v / v blocking donkey serum (Solarbio, catalog number #SL050), 2U / μL RNase Inhibitor (Xin Yichan, catalog number LS-EZ-E-00006P), 0.2 μg / mL DAPI, and the remainder is water. S8, Resuspend the MAP2-labeled cell nucleus sample obtained in S7 using 2 mL PBS; S9. The resuspended samples obtained in S8 were sorted using a flow cytometer to collect DAPI-positive cell nuclei (DAPI group) and MAP2 and DAPI double-positive cell nuclei (MAP2 group). The receiving solution was PBS with 0.1% BSA added w / v and RNase inhibitor at a final concentration of 2 U / μL.

[0020] A schematic diagram of the process of cell nuclear enrichment is shown below. Figure 1 As shown, the sorting results are as follows: Figure 2 As shown, the results indicate that the MAP2 positive rate in the MAP2 group was higher than that in the DAPI group. The formula for calculating the positive rate is: Positive rate = Number of MAP2 positive cell nuclei / Number of DAPI positive cell nuclei × 100%.

[0021] 2. Single-nuclear transcriptome sequencing of MAP2-highly expressing cells in mouse brain tissue S10, cell nuclei of the DAPI group and MAP2 group were stained with trypan blue dye and then counted; S11. Using the DNBelab C series high-throughput single-cell RNA library preparation kit (Shenzhen BGI Genomics Co., Ltd., catalog number 940-001818-00), DAPI and MAP2 libraries were constructed according to the kit instructions. S12, the constructed library was subjected to single-nucleus transcriptome sequencing (snRNA-seq) using the DNBSEQ-T7RS sequencing platform (Shenzhen BGI Genomics Co., Ltd.).

[0022] The results are as follows Figure 3 As shown, the results indicate that the mononuclear transcriptome library further constructed based on the cell nuclei enriched by the process in this embodiment has good signal-to-noise ratio and stability, meeting the requirements of subsequent high-throughput sequencing and downstream analysis.

[0023] Further comparison of sequencing data between the MAP2 group and the DAPI group yielded the following results: Figure 4 As shown, compared with the DAPI group, the proportion of neurons and excitatory neurons in the MAP2 group was significantly increased, while the proportion of oligodendrocytes and microglia with low Map2 expression levels was significantly decreased. Furthermore, the expression level of Map2 and the proportion of cells expressing it were significantly increased in the MAP2-positive group. This indicates that the flow cytometry sorting process in Example 1 can effectively enrich the target cell nuclei and achieve targeted acquisition of the cell population of interest.

[0024] The sequencing results of the MAP2 group were then compared with AllenBrain single-cell data (https: / / alleninstitute.github.io / abc_atlas_access / notebooks / getting_started.html) to analyze the differences in the proportion of mitochondrial genes. The results are as follows: Figure 5 As shown, compared with AllenBrain single-cell data obtained by traditional single-cell nucleus extraction methods, the sequencing data obtained in this embodiment retains more mitochondrial gene expression, that is, this process retains more cytoplasm.

[0025] Example 2: Nuclear enrichment and single-nuclear transcriptome sequencing of cells with high tyrosine hydroxylase (TH) expression in mouse brain tissue Referring to the steps described in Example 1, the difference is that: in step S7, the MAP2 marker is replaced with a TH marker diluted at a ratio of 1:400 (Santa Cruz, catalog number sc-25269); in step S9, DAPI-positive cell nuclei (DAPI group) and TH and DAPI double-positive cell nuclei (TH group) are collected; in step S16, the AccuNext single-cell / micro cDNA synthesis and amplification kit (Accurate Biotechnology, catalog number AG12501) and the AccuNext rapid DNA library preparation kit (Accurate Biotechnology, catalog number AG12522) are used for library construction, and the remaining steps remain unchanged.

[0026] The sequencing results were enriched using Gene Set Enrichment Analysis (GSEA), and the results are as follows: Figure 6 As shown, the method of the present invention can also enrich TH protein, which is also a cytoplasmic protein, in the GSEA results. TH Significant enrichment of genes.

[0027] Comparative Example 1: Nuclear enrichment and single-nuclear transcriptome sequencing of MAP2-highly-expressing cells in mouse brain tissue with altered procedures. Referring to the steps described in Example 1, the difference is that in step S1, DEPC in the lysis buffer TSTD solution is replaced with RNase Inhibitor at a final concentration of 2 U / μL, and step S5 is not performed, while steps S9 remain unchanged.

[0028] The full-length cDNA of the sorted DAPI-positive group was amplified using the AccuNext Single-Cell / Micro cDNA Synthesis and Amplification Kit (AccuNext Biotechnology, catalog number AG12501). The distribution characteristics of the full-length cDNA were detected using the Qsep400 Plus high-throughput nucleic acid and protein analysis system to determine RNA integrity, and the results were compared with those in Example 1. Figure 7 As shown, the cDNA main peak obtained by the method described in Example 1 was approximately 2125 bp, while the main peak obtained by the method described in Comparative Example 1 was only approximately 664 bp; this indicates that the method described in Example 1 can significantly improve RNA integrity and is beneficial for obtaining high-quality single-nucleus transcriptome libraries.

[0029] Comparative Example 2: Nuclear enrichment and single-nuclear transcriptome sequencing of MAP2-highly-expressing cells in mouse brain tissue with altered procedures. The steps described in Example 1 are the same, except that PBS is not added for washing in step S5, and the steps up to S8 remain unchanged.

[0030] Fluorescence signal analysis was performed using flow cytometry and compared with that in Example 1. The results are as follows: Figure 8 As shown, washing with PBS can effectively enhance the flow cytometry signal of fluorescent staining.

Claims

1. A method for enriching low-abundance cell nuclei in a sample, characterized by the following steps: include: (1) Lyse the tissue sample to obtain cell nuclei with the extranuclear cytoplasm retained; (2) The cell nuclei obtained in step 1 are labeled and sorted to obtain enriched low-abundance cell nuclei.

2. The method for enriching low-abundance cell nuclei in a sample according to claim 1, characterized in that, Step 1 includes: (11) The tissue sample was mixed with the lysis buffer and homogenized to obtain the lysis product; (12) Filter the lysate obtained in step 11, centrifuge and resuspend to obtain a cell nucleus suspension that retains the extranuclear cytoplasm.

3. The method for enriching low-abundance cell nuclei in a sample according to claim 2, characterized in that, The lysis buffer in step 11 contains 145-147 mM NaCl, 20-22 mM MgCl2, 9-11 mM Tris-HCl, 0.5-1.5 mM CaCl2, 0.008-0.012% w / v bovine serum albumin, 0.025-0.035% v / v polysorbate-20, a pharmacologically effective concentration of protease inhibitor, a pharmacologically effective concentration of RNase inhibitor, and the remainder is water; the homogenization step is performed using the type A pestle of a Dounce homogenizer.

4. The method for enriching low-abundance cell nuclei in a sample according to claim 2, characterized in that, The filtration step described in step 12 uses a sieve with a pore size of 35~45 μm for filtration; the centrifugation conditions are 2~8℃, 500×g centrifugation for 4~6 min.

5. The method for enriching low-abundance cell nuclei in a sample according to claim 1, characterized in that, Step 2 includes: (21) Fix the cell nuclei obtained in step 1 with the extranuclear cytoplasm retained, and wash them to obtain fixed cell nuclei; (22) Seal and fix the cell nucleus and label it; (23) The labeled cell nuclei obtained in step 22 are sorted to obtain enriched low-abundance cell nuclei.

6. The method for enriching low-abundance cell nuclei in a sample according to claim 5, characterized in that, The fixation step described in step 21 uses methanol for fixation; the washing step involves centrifuging at 2~8℃ and 500×g for 4~6 min, discarding the supernatant, resuspending in phosphate buffer, and centrifuging again at 2~8℃ and 500×g for 4~6 min, discarding the supernatant.

7. The method for enriching low-abundance cell nuclei in a sample according to claim 5, characterized in that, The blocking step in step 22 involves blocking at 2~8℃ for 10~20 min; the labeling step uses fluorescent antibodies and / or fluorescent probes for labeling.

8. The method for enriching low-abundance cell nuclei in a sample according to claim 5, characterized in that, Step 23 describes a sorting process using flow cytometry.

9. A cell nucleus, characterized in that, The cell nucleus is prepared by the method according to any one of claims 1 to 8.

10. The application of the cell nucleus as described in claim 9 in single-nucleus transcriptome sequencing.

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