Method for purification of nuclei for single-nucleus sequencing of frozen pig tissue and the resulting nuclei suspension and uses thereof

Abstract

The present application belongs to the technical field of flow cytometry sorting, and particularly relates to a method for purifying cell nuclei by flow cytometry sorting for single-nucleus sequencing of frozen pig tissues, cell nuclei suspension obtained by the method and application of the cell nuclei suspension. The present application introduces a flow cytometry gating and sorting step using 7-AAD as a nucleic acid dye on the basis of obtaining single-nucleus suspension by extracting cell nuclei from conventional frozen tissues. First, low scattering debris is excluded based on FSC / SSC parameters. Then, aggregate / doublet events are excluded based on FSC-A and FSC-H parameters. Finally, 7-AAD positive signals are detected in the PerCP-Cy5.5 channel to sort and obtain purified cell nuclei population. The flow cytometry sorting can effectively reduce cell debris and free nucleic acid background in the cell nuclei suspension, improve the purity of the single-nucleus suspension and the availability of subsequent single-nucleus transcriptome sequencing data, and is suitable for difficult samples with high cell debris and free nucleic acid background and low cell proportion.

Description

Technical Field

[0001] This invention belongs to the field of flow cytometry cell sorting technology, specifically relating to a flow cytometry method for purifying cell nuclei for single-cell nuclear sequencing of frozen porcine tissue, the resulting cell nucleus suspension, and its applications. Background Technology

[0002] Single-nucleus RNA sequencing (snRNA-seq) can be performed on frozen tissues or samples where intact live cells are difficult to obtain. In practice, the cell viability after thawing frozen tissues can be extremely low, even 0%, making single-cell protocols unsuitable and necessitating single-nucleus protocols. However, nuclear extraction from frozen tissues often results in a large amount of cell debris, impurities, and free RNA, leading to high background levels during library preparation and a low fractionation-in-cells (FIC) ratio.

[0003] Existing processes often rely on filtration, gradient centrifugation, or simple washing, which are insufficient to further improve the purity of cell nuclei in challenging samples where the nuclei are morphologically intact but still have a high background. To address these issues, a purification method is needed that can efficiently remove debris / impurities and reduce free nucleic acid background from cell nucleus suspensions without significantly damaging the cell nuclei. Summary of the Invention

[0004] The purpose of this invention is to address the sequencing data quality issues in porcine frozen tissue cell nucleus extraction samples, such as high levels of fragmented impurities, high background levels of cell debris and free nucleic acids, which may result in low fractional cell count (FIC) even after passing microscopic examination. This invention provides a flow cytometry method for single-cell nuclear sequencing of porcine frozen tissue, the resulting nuclear suspension, and its applications. This method improves the quality of the single-nucleus suspension and the usability of subsequent sequencing data by introducing a flow cytometry purification step.

[0005] According to a first aspect of the present invention, a method for nuclear flow cytometry sorting and purification for single-cell nuclear sequencing of frozen porcine tissue is provided, comprising the following steps: (1) Take frozen pig tissue samples, cut them at 0~4℃ and homogenize and lyse them to obtain a homogenate containing single cell nuclei; (2) The homogenate was filtered and centrifuged to enrich it, and then resuspended in a single-cell nucleus suspension buffer to obtain a single-cell nucleus suspension. (3) Add the nucleic acid fluorescent dye 7-Aminoactinomycin D (7-AAD, 7-aminoactinomycin D) to the single-cell nucleus suspension for staining; (4) The stained cell nucleus suspension was subjected to flow cytometry gating and sorting: the cell nucleus population was selected in the FSC / SSC scatter plot and low-scatter debris was excluded; single-particle events were screened in the FSC-A / FSC-H parameters and aggregates / duplexes were excluded; 7-AAD positive cell nucleus populations were selected in the 7-AAD fluorescence detection channel; In the FSC / SSC scatter plot, nuclear events were delineated based on their higher scattering signal compared to debris and their formation of relatively concentrated main clusters, while debris events with low FSC / low SSC were excluded. In the FSC-A / FSC-H scatter plot, single-particle events distributed along the main diagonal were selected, excluding aggregates or duplexes that deviated from this diagonal. Subsequently, 7-AAD-positive nuclear clusters were selected as the target sorting population in the 7-AAD fluorescence detection channel. (5) Collect the 7-AAD positive cell nuclei obtained in step (4) into a collection tube pre-cooled with collection solution to obtain a purified mononuclear suspension for subsequent mononuclear sequencing library construction.

[0006] This invention provides a flow cytometry-based method for nuclear sorting and purification in single-cell sequencing of frozen porcine tissue. Building upon conventional extraction of single-cell nuclei from frozen tissue, this method introduces a flow cytometry-gated and sorting step using 7-AAD as the nucleic acid dye. First, low-scattering debris is excluded based on FSC / SSC parameters. Then, aggregates / duplicate events are excluded based on FSC-A and FSC-H parameters. Finally, a 7-AAD positive signal is detected in the PerCP-Cy5.5 channel to obtain a high-purity nuclear population. This flow cytometry sorting effectively reduces cell debris and free nucleic acid background in the nuclear suspension, improving the purity of the single-cell nucleus suspension and the usability of subsequent single-cell nuclear transcriptome sequencing data. It is suitable for challenging samples with high cell debris and free nucleic acid background, and low cell percentage.

[0007] In some embodiments, the homogenization and lysis steps in step (1) are as follows: the cut frozen porcine tissue sample is placed in pre-cooled lysis buffer, and the tissue is gently ground or repeatedly blown around using a homogenizer to break it up, obtaining a tissue homogenate, which is then incubated with lysis buffer. The lysis buffer is a commercially available Nuclei EZ Lysis buffer (Sigma-Aldrich, NUC-101), supplemented with protease inhibitors (Roche, 5892791001) and RNase inhibitors (Promega, N2615; Life Technologies, AM2696) during use.

[0008] In some embodiments, the size of the cut pig frozen tissue sample in step (1) is 2±0.5 mm, and the homogenization is performed using a Dounce homogenizer in refrigerated lysis buffer, and the lysis / incubation is performed on ice.

[0009] In some implementations, step (2) filtering includes 40 Filter filtration with 20 The cell suspension was filtered to remove larger tissue fragments, insufficiently homogenized or incompletely dissociated clumps, and larger aggregates, thereby improving the homogeneity of the cell nucleus suspension. A 40 μm filter was used for initial filtration, and a 20 μm filter was used for further fine filtration.

[0010] In some embodiments, step (2) of the single-nucleus suspension buffer contains 1×PBS (phosphate-buffered saline), 0.07% w / v BSA (bovine serum albumin), and 0.1% v / v RNase inhibitor, wherein the PBS may be PBS with a pH of 7.2-7.4; the BSA may be bovine serum albumin used for preparing the nucleus suspension; and the RNase inhibitor may be an RNase inhibitor capable of inhibiting RNA degradation.

[0011] In some implementations, the amount of 7-AAD used in step (3) is 1 × 10 6 10 µL of each cell nucleus was incubated on ice in the dark for 4–6 min.

[0012] In some embodiments, the fluorescence detection channel for 7-AAD in step (4) is the PerCP-Cy5.5 channel or an equivalent 637~657 nm emission detection channel (other equivalent channels suitable for detecting 7-AAD fluorescence signals), with 647 nm being optimal. PerCP-Cy5.5 is a tandem fluorescent dye formed by coupling PerCP (polydinophyll chlorophyll protein complex) and Cy5.5 (cyanine dye).

[0013] In some implementations, the gating sequence of step (4) is as follows: first, defraction is performed according to FSC / SSC, then aggregates / duplexes are performed according to FSC-A / FSC-H, and finally, positive selection is performed using 7-AAD staining.

[0014] In some embodiments, the collected solution in step (5) is a pre-cooled buffer solution, and the entire process is carried out at 0-4°C or on ice. The pre-cooled buffer solution contains BSA and RNase inhibitors.

[0015] In some implementations, the cell viability of the frozen tissue sample is determined to be 0% by cell counting.

[0016] According to a second aspect of the present invention, a cell nucleus suspension obtained by the above-described flow cytometry purification method is provided, wherein the cell nucleus suspension has reduced fragmentation and background after flow cytometry separation, thus meeting the requirements for subsequent library construction.

[0017] According to a second aspect of the present invention, the above-described nuclear suspension is provided for use in single-nuclear transcriptome sequencing (snRNA-seq). For example, the high-purity nuclear suspension can be used for library preparation and loading for single-nuclear transcriptome sequencing, tissue and cell type identification, differential analysis of nuclear transcriptomes between different tissues or treatment conditions, and construction of tissue and cell atlases.

[0018] Compared to conventional cell nucleus extraction processes that rely solely on filtration / centrifugation, this invention achieves this through flow cytometry-gated sorting: (1) Effectively exclude low-scattering debris in the FSC / SSC dimension; (2) Exclude aggregate / dual cluster events in the FSC-A / FSC-H dimension to reduce the impact of nuclear aggregation on counting and library construction; (3) A purer cell nucleus population can be obtained through 7-AAD positive selection, thereby reducing background contamination from free nucleic acids and debris; (4) For samples with extremely low cell viability (preferably 0%) in frozen tissue, high-purity cell nuclei that can be used for library construction can still be obtained through sorting, thereby improving sample utilization.

[0019] This improves the uniformity and purity of single-cell nucleus suspensions, enhances the stability and reproducibility of subsequent library construction and instrumentation, and improves the applicability to difficult samples. Attached Figure Description

[0020] Figures 1A-1D This is a schematic diagram showing the counter results of a frozen porcine placental tissue sample L70-4-2. Figure 1B This is a diameter distribution map. Figure 1C This is a fluorescence intensity distribution map of FL1. Figure 1D This is a fluorescence intensity distribution diagram of FL2.

[0021] Figure 2This is a schematic diagram of flow cytometry-gated nuclear data for 7-AAD staining in porcine placental frozen tissue sample L70-4-2: including FSC / SSC defraction, FSC-A / FSC-H deaggregate / dipeptide removal, and PerCP-Cy5.5 channel 7-AAD positive selection; where A is an FSC-A / SSC-A scatter plot used to initially delineate the main nuclear population and exclude low-scatter debris; B is an FSC-A / FSC-H scatter plot used to screen for single-particle events and exclude aggregates / dipeptides; C is a PerCP-Cy5.5-A / SSC-A scatter plot used to select 7-AAD positive nuclear populations; D is a PerCP-Cy5.5-A / FSC-H scatter plot showing the distribution of the final target nuclear population in the overall event.

[0022] Figure 3 This is a schematic diagram of flow cytometry-gated 7-AAD staining of DN-3 frozen porcine muscle tissue samples: including FSC / SSC defraction, FSC-A / FSC-H deaggregate / dipeptide removal, and PerCP-Cy5.5 channel 7-AAD positive selection; where A is an FSC-A / SSC-A scatter plot used to initially delineate the main nuclear population and exclude debris; B is an FSC-A / FSC-H scatter plot used to screen for single-particle events and exclude aggregates / dipeptides; C is a PerCP-Cy5.5-A / SSC-A scatter plot used to select 7-AAD positive nuclear populations; D is a PerCP-Cy5.5-A / PerCP-Cy5.5-H scatter plot used for further fine screening of 7-AAD positive populations; E is a statistical table of the number and percentage of events in each gated population.

[0023] Figure 4 Microscopic image (10×) of the cell nuclei of a frozen porcine muscle tissue sample DN-3.

[0024] Figure 5 Microscopic image (20×) of the cell nuclei of a frozen porcine muscle tissue sample DN-3. Detailed Implementation

[0025] The present invention will be further described in detail below with reference to specific embodiments. It is worth noting that the following embodiments are only for better explaining the content of the present invention and do not limit the scope of protection of the present invention. Process steps not disclosed in the embodiments are prior art. Unless otherwise specified, all raw materials are commercially available.

[0026] This invention provides a combined scheme of "nuclear extraction + flow cytometry sorting and purification": first, a cell nucleus suspension is obtained by mild homogenization and lysis under low temperature conditions; then, a flow cytometry gated process is established using the staining signal of 7-AAD on the cell nucleus nucleic acid to separate and purify the cell nucleus from debris, aggregates and non-target particles, resulting in a high-purity cell nucleus suspension for library construction of single-nucleus transcriptome sequencing (snRNA-seq).

[0027] Example 1 I. Extraction of cell nuclei from frozen porcine tissue 1. Tissue processing and lysis Cell nuclei were isolated using Nuclei EZ lysis buffer (NUC-101; Sigma-Aldrich) supplemented with protease inhibitors (5892791001; Roche) and RNase inhibitors (N2615; Promega and AM2696; Life Technologies). Samples were cut into 2 mm pieces at 0–4 °C, homogenized in 2 mL of chilled Nuclei EZ lysis buffer using a Dounce homogenizer (885302-0002; Kimble Chase), and then lysed on ice for 5 min with 2 mL of lysis buffer added.

[0028] 2. Filtration and centrifugation Homogenization process 40 Cells were filtered through a cell filter (43-50040-51; pluriSelect) and then centrifuged at 5,000 g for 5 min at 4 °C. After centrifugation, the precipitate was resuspended, washed with 4 mL of lysis buffer, and lysed on ice for 5 min. After another centrifugation, the precipitate was resuspended in Nuclei Suspension Buffer (1x PBS, 0.07% BSA, and 0.1% RNase inhibitor) to form a nuclear suspension, and then lysed with 20 mL of lysate. Cellular filter (43-50020-50; pluriSelect) filtration.

[0029] II. 7-AAD staining and flow cytometry-gated purification 1. Staining Add 7-AAD to the cell nucleus suspension for staining. Example conditions: 10 µL / Test / 1 million cells, incubate on ice in the dark for about 5 min.

[0030] 2. Onboarding and Gate Control After establishing the flow cytometry template, the sample is loaded. First, the main nuclear population is delineated in the FSC / SSC scatter plot, and debris in the lower left corner is excluded; second, aggregates / duplexes are excluded in FSC-A / FSC-H; finally, 7-AAD positive nuclear populations are selected as target nuclei in the 7-AAD fluorescence detection channel (e.g., PerCP-Cy5.5).

[0031] 3. Collection 7-AAD positive cell nuclei were sorted into pre-cooled collection tubes. The collection solution could be a buffer containing BSA and RNase inhibitors. The entire process was kept at 4°C or on ice to reduce RNA degradation and nuclear structural damage.

[0032] Example 2: Nucleus extraction from frozen porcine placental tissue sample L70-4-2 This embodiment uses a frozen porcine placental tissue sample L70-4-2 as an example. The cell nucleus extraction procedure can be performed according to the steps described in Example 1. After obtaining a single-cell nucleus suspension, it is counted, evaluated, and sorted by flow cytometry.

[0033] Counting and evaluation: Sample L70-4-2 was analyzed using an AOPI counter. Example results showed a cell viability of 0%, a total cell count of 298, a live cell count of 0, a dead cell count of 298, an average roundness of approximately 0.91, a total cell concentration of approximately 4.45E+05 / mL, a live cell concentration of 0, a dead cell concentration of approximately 4.45E+05 / mL, a clumping rate of 0%, and an average diameter of approximately 6.42 mm. (See Figures 1A-1D This result suggests that live cells are difficult to obtain, making single-nucleus sequencing a more suitable approach.

[0034] Flow cytometry gating: After adding 7-AAD staining to the nuclear suspension, the cells are loaded onto the flow cytometer. The main nuclear populations are delineated using FSC / SSC, and debris is excluded. Aggregates / duplexes are then excluded using the FSC-A / FSC-H parameters. Finally, 7-AAD-positive nuclear populations are selected and collected using the PerCP-Cy5.5 channel (see...). Figure 2 ).

[0035] like Figure 2 As shown, after initial screening by FSC / SSC, sample L70-4-2 can obtain a main nuclear population that can be distinguished from low-scattering debris; further, single-particle events distributed along the main diagonal are selected in the FSC-A / FSC-H scatter plot and aggregates / duplexes are excluded; then, 7-AAD positive nuclear populations are selected as the target sorting population in the PerCP-Cy5.5 channel.

[0036] Example 3: Nuclear extraction and results of DN-3 cells from frozen porcine muscle tissue samples. This embodiment uses a frozen porcine muscle tissue sample DN-3 as an example. The cell nucleus extraction procedure can be performed according to the steps described in Example 1. After obtaining a single-cell nucleus suspension, flow cytometry gating sorting and microscopic examination are performed.

[0037] Flow cytometry gating: After adding 7-AAD staining to the nuclear suspension, the cells are loaded onto the flow cytometer. The main nuclear populations are delineated using FSC / SSC, and debris is excluded. Aggregates / duplexes are then excluded using the FSC-A / FSC-H parameters. Finally, 7-AAD-positive nuclear populations are selected and collected using the PerCP-Cy5.5 channel (see...). Figure 3 ).like Figure 3 As shown, after FSC / SSC gating, the main nuclear population and low-scattering debris were clearly distinguishable in sample DN-3. Further analysis of the FSC-A / FSC-H diagrams revealed single-particle events distributed diagonally, excluding aggregates / duplexes. Subsequently, 7-AAD-positive nuclei were selected as target nuclei in the PerCP-Cy5.5 channel and sorted. Microscopic examination after sorting showed that the obtained nuclei were morphologically intact, well-dispersed, and without significant large-scale aggregation, meeting the requirements for subsequent mononuclear transcription library construction.

[0038] Microscopic examination: After sorting, the cell nuclei should be examined under a microscope. The nuclei should be morphologically intact, without obvious overlap or large-scale aggregation, and uniformly distributed. Figure 4 The image shows the results of a 10x microscopic examination of the sample. Figure 5 The image shows the results of a 20x magnification examination of the sample.

[0039] The above descriptions are merely some specific embodiments of the present invention. For those skilled in the art, various modifications and improvements can be made without departing from the inventive concept of the present invention, and all such modifications and improvements fall within the scope of protection of the present invention.

Claims

1. A method for nuclear flow cytometry sorting and purification for single-cell nuclear sequencing of frozen porcine tissue, characterized in that, Includes the following steps: (1) Take frozen pig tissue samples, cut them at 0~4℃ and homogenize and lyse them to obtain a homogenate containing single cell nuclei; (2) The homogenate was filtered and centrifuged to enrich it, and then resuspended in a single-cell nucleus suspension buffer to obtain a single-cell nucleus suspension. (3) Add nucleic acid fluorescent dye 7-AAD to the single cell nucleus suspension for staining; (4) The stained cell nucleus suspension was subjected to flow cytometry gating and sorting: cell nuclei were selected and debris was excluded from the FSC / SSC scatter plot; aggregates / duplexes were excluded from the FSC-A / FSC-H parameters; 7-AAD positive cell nuclei were selected from the 7-AAD fluorescence detection channel; (5) Collect the 7-AAD positive cell nuclei obtained in step (4) into a collection tube pre-filled with collection solution to obtain a single-nuclear suspension for subsequent single-nuclear sequencing library construction.

2. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 1, characterized in that, In step (1), the homogenization and lysis steps are as follows: the cut frozen pig tissue sample is placed in pre-cooled lysis buffer, and the tissue is gently ground or repeatedly blown and broken up using a homogenizer to obtain a tissue homogenate, which is then incubated with lysis buffer.

3. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 2, characterized in that, The lysis buffer is Nuclei EZ Lysis buffer, with added protease inhibitors and RNase inhibitors.

4. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 1, characterized in that, Step (2) filtration includes filtration with a 40 μm filter and filtration with a 20 μm filter.

5. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 1, characterized in that, In step (2), the single-cell nucleus suspension buffer contains 1×PBS, 0.07% w / v BSA and 0.1% v / v RNase inhibitor.

6. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 1, characterized in that, The dosage of 7-AAD in step (3) is 1 × 10 6 10 µL per cell nucleus.

7. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 1, characterized in that, The fluorescence detection channel of the 7-AAD mentioned in step (4) is the PerCP-Cy5.5 channel or its equivalent 637~657 nm emission detection channel.

8. The method for nuclear flow cytometry sorting and purification for single-cell sequencing of frozen porcine tissue according to claim 1, characterized in that, In step (5), the collection solution is a pre-cooled buffer solution.

9. A cell nucleus suspension obtained using the cell nucleus flow cytometry sorting and purification method for single-cell sequencing of frozen porcine tissue as described in any one of claims 1-8.

10. Use of the nuclear suspension of claim 9 in mononuclear transcriptome sequencing.

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