RNA (Ribonucleic Acid) and DNA (Deoxyribose Nucleic Acid) dual-fluorescence activated cell sorting technique and application thereof in cell subtype typing and cell genetic material research

Abstract

The invention provides an RNA (Ribonucleic Acid) and DNA (Deoxyribose Nucleic Acid) dual-fluorescence activated cell sorting technique and application thereof in cell subtype typing and cell genetic material research, and belongs to the technical field of molecular biology, the RNA and DNA dual-fluorescence activated cell sorting technique comprises the following steps: 1) digesting cell tissues to obtain a single-cell suspension; 2) carrying out double staining of DNA and RNA on the digested single-cell suspension; and (3) carrying out flow cytometry analysis and fluorescence activation cell sorting on the dyed cells. According to the method, RNA and DNA in the cells are subjected to double staining, so that cell types which are difficult to distinguish in the past can be distinguished more accurately. The invention provides a Ramp; the D-FACS provides an effective means for further fine research on the spermatogenesis process, and provides an effective method for research on sperm abnormality and sterility.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular biology, and in particular relates to RNA and DNA dual fluorescence activated cell sorting and its application in cell subtype typing and cell genetic material research. Background Art

[0002] The testis is known for its highly complex gene expression profile, with more than 80% of protein-coding genes transcribed in this organ. The testis exhibits the most diverse types of genes that change synchronously with spermatogenesis (Brawand, D., Soumillon, M., Necsulea, A., Julien, P., Csárdi, G., Harrigan, P., Weier, M., Liechti, A., Aximu-Petri, A., Kircher, M., et al. (2011). The evolution of gene expression levels in mammalian organs. Nature 478, 343-348.), but it is unclear whether the total amount of testicular RNA also fluctuates accordingly. Although the transcriptional diversity of germ cells has been widely studied, the dynamic changes in RNA abundance at different stages and their biological significance remain little explored. Accurate cell subtyping is the basis for studying the role of inherited substances (DNA, RNA and protein) and can provide important research basis for many diseases. Currently, cell subtyping mainly uses flow cytometry, but there is a lack of accurate cell subtyping methods for DNA and / or RNA. Summary of the invention

[0003] In view of this, the purpose of the present invention is to provide an RNA and DNA dual fluorescence activated cell sorting method and its application in cell subtyping and cell genetic material research. The method provided by the present invention not only expands the capabilities of traditional Hoechst-based FACS and achieves the separation of known cell populations, but also can accurately classify cell types that were previously difficult to distinguish, such as pachytene spermatocytes, diplotene spermatocytes, round spermatocytes and elongated spermatocytes. The present invention also successfully distinguishes two MII cell subtypes and identifies cells in the first meiotic division (MI) M phase.

[0004] The present invention provides a dual fluorescence activated cell sorting method for RNA and DNA, comprising the following steps:

[0005] 1) Digest the cell tissue to obtain a single cell suspension;

[0006] 2) Double staining of DNA and RNA in the digested single cell suspension;

[0007] 3) Perform flow cytometry analysis and fluorescence activated cell sorting on the stained cells;

[0008] The DNA staining solution is Hoechst 33342, and the RNA staining solution is RNASelect TM Green fluorescent cell stain.

[0009] Preferably, the double staining in step 2) is to mix and incubate the DNA staining solution Hoechst 33342, the RNA staining solution SYTOgreen and the single cell suspension.

[0010] Preferably, the incubation temperature is 36-38° C., and the incubation time is 25-35 min.

[0011] Preferably, the amount of the DNA staining solution Hoechst 33342 is 3 to 8 μg / 10 6 cells; the RNA staining solution is RNASelect TM The dosage of green fluorescent cell dye is 450~550nmol / 10 6 cells.

[0012] The present invention also provides the application of the RNA and DNA dual fluorescence activated cell sorting technique in cell subtype typing.

[0013] Preferably, the cell subtyping includes spermatogenic cell subtyping.

[0014] Preferably, the spermatogenic cell subtyping classifies testicular single cells into the following groups: differentiated spermatogonia, Pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, Pachytene spermatocytes, Diplotene spermatocytes, MI spermatocytes, MII spermatocytes, round spermatids, elongated spermatids and Sertoli supporting cells.

[0015] The invention also provides the application of the RNA and DNA dual fluorescence activated cell sorting technique in the study of cell genetic material.

[0016] Preferably, the genetic material studies include changes in RNA abundance.

[0017] The present invention also provides the application of the RNA and DNA dual fluorescence activated cell sorting technique in the functional research of RNA binding protein.

[0018] Compared with the prior art, the present invention has the following beneficial effects: the present invention provides an RNA and DNA dual fluorescence activated cell sorting technique (hereinafter referred to as R&D-FACS) and its application in cell subtyping and cell genetic material research. The present invention can effectively distinguish the characteristics of DNA and RNA changes in different stages of cells by double staining RNA and DNA in cells, and can more accurately distinguish different cell subtypes, especially germ cells with richer dynamic changes in RNA abundance.

[0019] Furthermore, the R&D-FACS provided by the present invention can also study the function of RNA binding proteins, and the role of RNA binding protein DDX17 in spermatogenesis was studied by R&D-FACS. After DDX17 knockout (KO), significant abnormal changes in RNA abundance in the pre-L stage were observed. DDX17 plays a key role in regulating transcription and post-transcriptional events in the process of spermatogenesis. The loss of DDX17 disrupts RNA transcription and alternative splicing, leading to DSB repair and homologous chromosome synapsis defects, and ultimately leading to spermatogenesis failure and male infertility; the R&D-FACS provided by the present invention provides an effective means for further fine-tuning the process of spermatogenesis, and provides an effective method for subsequent studies of sperm abnormalities and infertility. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 Figure 2. Flow cytometric analysis of adult mouse testicular cells using different staining methods, where (A) debris was excluded based on low light scatter parameters, and FSC and SSC, which are proportional to cell size and granularity, were used to distinguish cells from debris, and flow cytometric analysis of adult mouse testicular cells based on synthetic green fluorescence showed a FITC histogram; (B) flow cytometric fluorescence image of adult mouse testicular cells co-stained with Hoechst and SYTOgreen, and cells were visible in the Hoechst blue / Hoechst red color map; (C) flow cytometric analysis of adult mouse testicular cells based on Hoechst fluorescence, shown in the FITC histogram and Hoechst blue / Hoechst red pseudocolor map; (D) flow cytometric analysis of adult mouse testis, cells using two staining schemes: co-stained with SYTOred and SYTOgreen, and triple stained with Hoechst, SYTOred and SYTOgreen, and cells were visible in the FITC and Cy5.5 histograms, Hoechst blue / Hoechst red pseudocolor map, and Hoechst blue / FITC pseudocolor map.

[0021] Figure 2The construction and application of R&D-FACS based on SYTOgreen dye, including A) SYTOgreen (green) immunohistochemical staining of adult wild-type mouse testis cryosections; scale bar = 20 μm; the detailed spermatogenic cell composition is framed and marked in the enlarged image; B) SYTOgreen fluorescent staining of adult mouse testis cryosections after RNaseA and RNase H treatment, proving that it is specific RNA staining; scale bar = 20 μm; C) flow cytometric fluorescence image of adult mouse testis cells co-stained with Hoechst and SYTOgreen; cells are visualized in Hoechst blue / Hoechst red and Hoechst blue / FITC pseudocolor images; P1-P9 represent nine stable and distinguishable cell populations in the fluorescence image, as shown in the schematic diagram of the fluorescence image in the figure.

[0022] Figure 3 Identification of subpopulations in R&D-FACS fluorescence maps, where A) the Hoechst blue / FITC fluorescence map generated by R&D-FACS shows nine different subpopulations, merged into the Hoechst blue / Hoechst red map, and the blue markers indicate the corresponding positions of these subpopulations on the Hoechst blue / Hoechst red map; B) the workflow of low-input RNA-seq, outlining how to extract RNA from sorted subpopulations for sequencing; C) a heat map of selected spermatogenic cell marker genes, showing different expression features in the nine subpopulations; D) Pearson correlation analysis of RNA expression profiles between the nine subpopulations, illustrating the relationship between these subpopulations; E) a heat map showing the correlation between bulk RNA-seq data obtained from sorted testicular cells and the pseudo-bulk RNA-seq gene expression matrix obtained from single-cell transcriptomics.

[0023] Figure 4To verify the spermatogenic subpopulations and their RNA expression profiles using capillary electrophoresis; A) Chromosome spreads of P1 and P2 cells co-stained with γ-H2AX (green), which marks double-strand breaks [DSBs], and SYCP3 (red), which marks the axial element. P1 is mainly composed of diplotene spermatocytes, while P2 is composed of pachytene spermatocytes. Scale bar = 5 μm; B) DAPI staining of P6 and P9 subpopulations, showing chromosome distribution, scale bar = 10 μm; C) Heat map of total RNA of the nine subpopulations obtained by R&D-FACS, measured by capillary electrophoresis at intervals of 200 nucleotides, and the color gradient from white to red indicates the proportion of RNA of different lengths, representing the increase in RNA abundance; D) The proportion of rRNA in P4 (diplotene spermatocytes [D]) and P3 (pachytene spermatocytes [P]) subpopulations, calculated from the merged 5.8S, 18S, and 28S peaks. Data are presented as mean ± SEM based on three biological replicates, two-tailed Student's t test: P < 0.01; E) Representative capillary electrophoresis RNA profiles of P7 (meiosis II spermatocyte 2 [MII2]) and P8 (round spermatids [RS]) subpopulations, showing high similarity in RNA distribution and ratio.

[0024] Figure 5Knockout of DDX17 leads to infertility in mice and causes abnormalities in homologous chromosome synapsis and DNA double-strand break repair; A) Testicular morphological analysis showed that the testes of DDX17 knockout mice (DDX17- / -,stra8-cre) were significantly smaller than those of the corresponding littermate control mice (DDX17wt); the bar graph shows the comparison of testicular weight between 8-week-old DDX17 knockout mice and age-matched control mice; Student's t-test was used for statistical analysis, and the data are the mean ± SEM of ten independent experiments, P < 0.01, B) Hematoxylin and eosin (HE) staining of the testes and epididymis of DDX17 knockout mice and control mice; scale bar = 100 μm; C) TUNEL staining shows apoptotic cells in the testes of DDX17 knockout mice and control mice, and the bar graph shows the number of TUNEL-positive cells in the seminiferous tubules; Student's t-test was used for statistical analysis, and the data are the mean ± SEM of ten independent experiments, P < 0.01, B) Hematoxylin and eosin (HE) staining of the testes and epididymis of DDX17 knockout mice and control mice; scale bar = 100 μm; C) TUNEL staining shows apoptotic cells in the testes of DDX17 knockout mice and control mice, and the bar graph shows the number of TUNEL-positive cells in the seminiferous tubules; Student's t-test was used for statistical analysis. Statistical analysis was performed using t-test, and data are the mean ± SEM of five independent experiments, P < 0.01; D) Immunofluorescence staining of DDX17 in the testes of DDX17 knockout mice and control mice; DNA was stained with DAPI; E) R&D-FACS fluorescence images of testicular cells of DDX17 knockout mice and control wild-type (WT) mice, with significant differences highlighted in red; RNA abundance increased in knockout mice at the pre-leptotene (pre-L) stage and matched that of the control group at the leptotene / zygotene (L / Z) stage; F) Immunofluorescence staining of SYCP3 and SYCP1 in spermatocytes of DDX17WT and DDX17- / -, stra8-cre mice; Scale bar = 5 μm; G) Immunofluorescence staining of SYCP3 and γH2AX in spermatocytes of DDX17WT and DDX17- / -, stra8-cre mice, Scale bar = 5 μm.

[0025] Figure 6Figure 3. DDX17 knockout leads to transcriptional dysregulation and abnormal alternative splicing events; A) Volcano plot shows the number of genes (DEGs) that are significantly differentially expressed between DDX17 knockout mice and wild-type mice at the pre-leptotene (pre-L) and leptotene / zygotene (L / Z) stages. The purple dots on the left represent genes that are significantly down-regulated in DDX17cKO compared with DDX17WT, the blue dots on the right represent genes that are significantly up-regulated, and the gray dots represent genes with no significant differential expression; B) Heat map of the three main functional categories of selected key genes, showing the different expression characteristics between DDX17WT and DDX17cKO mice at pre-leptotene and leptotene / zygotene cells. Signs, the color scale from red to blue indicates the gene expression level from high to low; C) GO enrichment analysis, including biological process (BP), cellular component (CC), and molecular function (MF), showed the potential functions of transcripts that were significantly down-regulated in DDX17WT and DDX17cKO mice in proleptotene and leptotene / zygotene stages; selection criteria: p value ≤ 0.05; D) Splicing event information of genes related to DSB repair, recombination, and synapsis that were significantly different between DDX17WT and DDX17cKO mice in proleptotene and leptotene / zygotene cells; Fisher's exact test was used to identify significant splicing events between the two groups, ** indicates p value ≤ 0.05. DETAILED DESCRIPTION

[0026] The invention provides a dual fluorescence activated cell sorting method for RNA and DNA, comprising the following steps: 1) digesting cell tissue to obtain a single cell suspension; 2) double staining DNA and RNA on the digested single cell suspension; 3) performing flow cytometry analysis and fluorescence activated cell sorting on the stained cells; the DNA staining solution is Hoechst 33342, and the RNA staining solution is SYTO green.

[0027] In the present invention, cell tissue is digested to obtain a single cell suspension; the digestion solution used for the digestion includes a first digestion solution and a second digestion solution, the first digestion solution is preferably a Dulbecco's phosphate buffer containing type I collagenase and DNase I, in the first digestion solution, the concentration of type I collagenase is preferably 150-250U / mL, more preferably 200U / mL, the concentration of DNase I is preferably 4-6mg / mL, more preferably 5mg / mL; the temperature of the first digestion is 36-38°C, the time of the first digestion is 10-20min, the second digestion solution is preferably a trypsin solution, the concentration of the trypsin solution is preferably 0.04-0.06%, more preferably 0.05%. The temperature of the second digestion is 36-38°C, and the time of the second digestion is 10-20min. After the digestion, the present invention centrifuges and discards the supernatant, and resuspends the cells in PBS to obtain a cell suspension. In the present invention, the centrifugal force of the centrifugation is preferably 450-550×g, more preferably 500×g, the centrifugal time is preferably 4-6 min, more preferably 5 min; the centrifugal temperature is preferably 3-5°C, more preferably 4°C.

[0028] After obtaining the single cell suspension, the present invention performs double staining of DNA and RNA; the double staining is to mix and incubate the single cell suspension with the DNA staining solution Hoechst 33342, the RNA staining solution SYTO green. In the present invention, the incubation temperature is preferably 36 to 38°C, more preferably 37°C, and the incubation time is preferably 25 to 35 minutes, more preferably 28 to 32 minutes. In the present invention, the dosage of the DNA staining solution Hoechst 33342 is preferably 3 to 8 μg / 10 6 cells, more preferably 4 to 7 μg / 10 6 cells, the most preferred is 5 μg / 10 6 The amount of the RNA staining solution SYTO green is preferably 500nmol / 10 6 cells.

[0029] The present invention performs flow cytometry analysis and fluorescence activated cell sorting after the double staining. In the present invention, the cell suspension after the double staining is preferably filtered with a 70 μm cell sieve for flow cytometry analysis and fluorescence activated cell sorting (FACS). In the present invention, flow cytometry and cell sorting analysis are performed using the FACS Aria II flow cytometer of BD Biosciences and the MoFLOXDP high-speed flow cytometer sorting system of BeckmanCoulter. Different cell populations are distinguished according to fluorescence intensity. The laser setting includes a 488nm laser for forward scatter (FSC-A) and side scatter (SSC-A) detection, a 355nm ultraviolet laser for Hoechst 33342 excitation, a 450 / 40nm bandpass filter for detecting "Hoechst blue" signals, and a 670nm longpass filter for detecting "Hoechst red" signals. SYTO green dye is detected at an absorption / emission wavelength of 490 / 530nm through the FITC channel. Cell sorting used a 70 μm nozzle and the sorting rate was maintained at 4000 to 5000 events per second.

[0030] The present invention also provides the application of the RNA and DNA dual fluorescence activated cell sorting technique in cell subtype typing.

[0031] In the present invention, the cell subtyping includes spermatogenic cell subtyping.

[0032] The present invention also provides the application of the RNA and DNA dual fluorescence activated cell sorting technique in the study of cellular genetic material; preferably, the genetic material study includes changes in RNA abundance.

[0033] The present invention also provides the application of the RNA and DNA dual fluorescence activated cell sorting technique in the functional research of RNA binding protein.

[0034] The technical solutions provided by the present invention are described in detail below in conjunction with the embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0035] Example 1

[0036] Determination of RNA and DNA dual fluorescence activated cell sorting (R&D-FACS) staining solution and detection of staining effect

[0037] Test method:

[0038] 1. RNA-specific dyes RNASelect TM Flow cytometric analysis of testicular cells using a green fluorescent cell stain

[0039] Mice were killed by cervical dislocation and the testicles were removed by dissection. After removing the tunica albuginea, the testicles were placed in 5 mL Dulbecco's phosphate buffered saline (DPBS) containing 200 U / mL collagenase type I (Thermo Fisher Scientific, 17100017) and 5 mg / mL DNase I (Sigma-Aldrich, DN25) and incubated at 37°C for 15 minutes until the seminiferous tubules were completely dispersed and precipitated. The supernatant was removed and the precipitated tubules were resuspended in 20 mL 0.05% trypsin (Gibco, 25200072) and digested at 37°C for another 15 minutes. The digestion was terminated by adding 30 mL Dulbecco's modified Eagle's medium (DMEM) (Gibco, C11995500BT) containing 10% fetal bovine serum (FBS). After terminating the digestion, the subsequent staining operation was carried out directly without washing.

[0040] Add 500 nM to 10 ml of cell suspension RNASelect TM Green fluorescent cell stain (Thermo Fisher Scientific) was added, mixed gently, and incubated at 37°C for 30 minutes. Subsequently, the cells were centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in phosphate-buffered saline (PBS) and filtered through a 70 μm cell strainer (Corning, 352340) for flow cytometry analysis.

[0041] Flow cytometry and cell sorting analyses were performed using a FACS Aria II flow cytometer from BD Biosciences and a MoFLOXDP high-speed flow cytometric sorting system from Beckman Coulter. RNASelect TM Green fluorescent dye was detected through the FITC channel at an absorption / emission wavelength of 490 / 530 nm. Cell sorting used a 70 μm nozzle and the sorting rate was maintained at 4000–5000 events per second.

[0042] 2. Staining Experiments on Cryosections of Wild-type Mouse Testes

[0043] Male wild-type mice were killed by cervical dislocation, and testicular and epididymal tissues were quickly removed. Tissue samples were fixed in Bouin's solution at 4°C for 24 h for subsequent histological analysis. After fixation, the samples were transferred to 4% paraformaldehyde solution (Sigma-Aldrich, 158127-500G) at 4°C and fixed overnight in preparation for immunostaining. After a series of ethanol washes and dehydration, the samples were embedded in paraffin and sectioned using a Leica microdissection machine (Leica Biosystems, Germany). After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (H&E) for histological evaluation.

[0044] 3. RNaseA and RNase H treatment of SYTO green staining

[0045] SYTOgreen fluorescence staining of mouse testis cryosections after treatment with RNaseA and RNase H.

[0046] This experiment used C57BL / 6 wild-type male mice (8-12 weeks old) and was humanely euthanized by cervical dislocation. The mouse testicles were removed and immediately placed in OCT cryopreservation medium and frozen at -80°C. Subsequently, the testicular tissue was cut into 10-12 μm thick frozen sections using a cryostat (Leica CM1950), and the sections were fixed on slides for subsequent processing.

[0047] To remove single-stranded RNA and R-loop structures, two sections were used: one section was treated with RNase A and the other with RNase H. First, one section was incubated with 10 μg / mL RNaseA (Yeasen, Cat#10405ES03) at 37°C for 30 minutes to remove single-stranded RNA. After treatment, the sections were washed thoroughly with RNase-free PBS buffer. The other section was incubated with 2U / μL RNase H (Yeasen, Cat#12906ES60) at 37°C for 30 minutes to remove RNA in R-loops. After each enzyme treatment, the sections were thoroughly washed with RNase-free PBS buffer.

[0048] After enzyme treatment, two sections were stained with SYTOgreen fluorescence. SYTOgreen dye (ThermoFisher Scientific, Cat# S32703) was applied to the sections at a working concentration of 500 nM and incubated at room temperature for 20 minutes. After incubation, the sections were washed with PBS for 5 minutes and washed twice to remove excess dye.

[0049] For immunofluorescence staining, the sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 15 minutes after dewaxing and rehydration, and then cooled to room temperature. The sections were washed with PBS containing 0.1% TritonX-100 (PBST) and blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Subsequently, the sections were incubated with the primary antibody overnight at 4°C. After washing three times with PBST, the sections were incubated with the secondary antibody and 4,6-diamidino-2-phenylindole (DAPI) at 2°C for 1 hour. Finally, the sections were washed and mounted, and the samples were observed using a fluorescence microscope (Carl ZeissAG, LSM710, Germany). The antibodies used were as follows: DDX17 rabbit polyclonal antibody (1:200 dilution; Proteintech, 19910-1-AP), and TUNEL staining was performed according to the manufacturer's instructions (KeyGEN BioTECH, KGA7072).

[0050] 4.DNA dye Hoechst 33342 and RNASelect TM Green fluorescent cell stain combined with double staining

[0051] Hoechst 33342 (5 μg / 10 6 cells) (Thermo Fisher Scientific) and 500 nM / 10 6 Cells RNASelect TM Green fluorescent cell stain (ThermoFisher Scientific), gently mixed, and incubated at 37°C for 30 minutes. Subsequently, centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in phosphate-buffered saline (PBS) and filtered through a 70 μm cell strainer (Corning, 352340) for flow cytometry analysis and fluorescence-activated cell sorting (FACS).

[0052] Flow cytometry and cell sorting analysis were performed using a BD Biosciences FACS Aria II flow cytometer and Beckman Coulter MoFLOXDP high-speed flow cytometry sorting system. Staining protocols included the use of Hoechst 33342 or Hoechst 33342 / SYTO TMDifferent combinations of green fluorescent nucleic acid dyes distinguish different cell populations based on fluorescence intensity. The laser settings include a 488nm laser for forward scatter (FSC-A) and side scatter (SSC-A) detection, a 355nm UV laser for Hoechst 33342 excitation, a 450 / 40nm bandpass filter for detecting "Hoechst blue" signals, and a 670nm longpass filter for detecting "Hoechst red" signals. RNASelect TM Green fluorescent cell stain was detected via the FITC channel at an absorbance / emission wavelength of 490 / 530 nm. Cell sorting was performed using a 70 μm nozzle and the sorting rate was maintained at 4000–5000 events per second.

[0053] 5. DNA dye SYTO Deep Red (SYTO red) and RNASelect TM Green fluorescent cell stain combined with double staining

[0054] The operation steps are the same as 4, the only difference is that SYTO Deep Red is added.

[0055] Test results:

[0056] 1. Flow cytometry analysis of testicular cells was performed using SYTOgreen, a dye specific for RNA. The results showed that a wide range of fluorescence intensity was observed in the FITC channel ( Figure 1 A)

[0057] 2. Further staining experiments on frozen sections of wild-type mouse testes revealed that the fluorescence intensity of SYTO green varied in different testicular cell types ( Figure 2 A).

[0058] 3. The present invention also found RNASelect TM The fluorescence signal of the green fluorescent cell stain disappeared completely after RNaseA treatment, while RNase H treatment had no significant effect ( Figure 2 B in Figure 1). This confirms that SYTOgreen specifically binds to intracellular RNA and does not bind to other structures such as RNA-DNA hybrids, thus verifying its reliability as a marker of RNA abundance. The different fluorescence spectra observed indicate that the total amount of cellular RNA fluctuates significantly during spermatogenesis.

[0059] 4. Use only RNASelect TMGreen fluorescent cell staining to label RNA is not sufficient to distinguish different types of spermatogenic cells. To further improve the accuracy of cell classification, the DNA dye Hoechst 33342 was used in combination with RNASelect TM Green fluorescent cell stain is combined for dual staining. These two fluorescent dyes do not interfere with each other in terms of spectral characteristics and staining targets ( Figure 1 Unique fluorescence patterns in the Hoechst blue channel and the FITC channel enable precise classification of testicular cells into nine different populations based on their DNA and RNA content ( Figure 2 C). By using this method, RNA features related to changes in DNA content can be observed, confirming that changes in RNA content and DNA content during spermatogenesis are dynamically parallel.

[0060] 5. To further explore the potential of using other dyes to optimize the differentiation of different cell populations, triple co-staining was performed with SYTO DeepRed (red fluorescence), SYTO green (green fluorescence) and Hoechst (excitation light is blue). Unexpectedly, SYTO red staining is incompatible with Hoechst staining. Specifically, during triple staining, cells preferentially absorbed Hoechst 33342, indicating that both Hoechst 33342 and SYTO red bind to the same DNA targets and that Hoechst 33342 has a higher affinity for these DNA sites than SYTO red ( Figure 1 D).

[0061] Example 2

[0062] Refinement of spermatogenic cell subsets based on RNA abundance using R&D-FACS

[0063] Test method:

[0064] Isolation of spermatocytes

[0065] Mice were killed by cervical dislocation and the testicles were removed by dissection. After removing the tunica albuginea, the testicles were placed in 5 mL Dulbecco's phosphate buffered saline (DPBS) containing 200 U / mL collagenase type I (Thermo Fisher Scientific, 17100017) and 5 mg / mL DNase I (Sigma-Aldrich, DN25) and incubated at 37°C for 15 minutes until the seminiferous tubules were completely dispersed and precipitated. The supernatant was removed and the precipitated tubules were resuspended in 20 mL 0.05% trypsin (Gibco, 25200072) and digestion was continued at 37°C for 15 minutes. The digestion was terminated by adding 30 mL Dulbecco's modified Eagle's medium (DMEM) (Gibco, C11995500BT) containing 10% fetal bovine serum (FBS).

[0066] Flow cytometric analysis and fluorescence-activated cell sorting

[0067] Hoechst 33342 (5 μg / 10 6 cells) (Thermo Fisher Scientific) and 500 nM / 10 6 Cells RNASelect TM Green fluorescent cell stain (ThermoFisher Scientific), gently mixed, and incubated at 37°C for 30 minutes. Subsequently, centrifuged at 500×g for 5 minutes at 4°C, and the supernatant was discarded. The cells were resuspended in phosphate-buffered saline (PBS) and filtered through a 70 μm cell strainer (Corning, 352340) for flow cytometry analysis and fluorescence-activated cell sorting (FACS).

[0068] Flow cytometry and cell sorting analysis were performed using a BD Biosciences FACS Aria II flow cytometer and Beckman Coulter MoFLOXDP high-speed flow cytometry sorting system. Staining protocols included the use of Hoechst 33342 or Hoechst 33342 / RNASelect TMDifferent combinations of green fluorescent cell stains distinguish different cell populations based on fluorescence intensity. The laser settings included a 488 nm laser for forward scatter (FSC-A) and side scatter (SSC-A) detection, a 355 nm UV laser for Hoechst33342 excitation, a 450 / 40 nm bandpass filter for detecting "Hoechst blue" signals, and a 670 nm longpass filter for detecting "Hoechst red" signals. SYTO dye was detected through the FITC channel at an absorption / emission wavelength of 490 / 530 nm. Cell sorting used a 70 μm nozzle and the sorting rate was maintained at 4000–5000 events per second.

[0069] Histology and immunostaining

[0070] Male mice were killed by cervical dislocation, and testicular and epididymal tissues were quickly removed. Tissue samples were fixed in Bouin's solution at 4°C for 24 hours for subsequent histological analysis. After fixation, the samples were transferred to 4% paraformaldehyde solution (Sigma-Aldrich, 158127-500G) at 4°C and fixed overnight in preparation for immunostaining. After a series of ethanol washes and dehydration, the samples were embedded in paraffin and sectioned using a Leica microdissection machine (Leica Biosystems, Germany). After dewaxing and rehydration, the sections were stained with hematoxylin and eosin (H&E) for histological evaluation.

[0071] For immunofluorescence staining, sections were boiled in 10 mM sodium citrate buffer (pH 6.0) for 15 minutes after dewaxing and rehydration, and then cooled to room temperature. Sections were washed with PBS containing 0.1% Triton X-100 (PBST) and blocked with 5% bovine serum albumin (BSA) for 1 hour at room temperature. Subsequently, sections were incubated with primary antibodies overnight at 4°C. After washing three times with PBST, sections were incubated with secondary antibodies and 4,6-diamidino-2-phenylindole (DAPI) at 2°C for 1 hour. Finally, washing and mounting were performed, and samples were observed using a fluorescence microscope (Carl Zeiss AG, LSM710, Germany). The antibodies used were as follows: DDX17 rabbit polyclonal antibody (1:200 dilution; Proteintech, 19910-1-AP), and TUNEL staining was performed according to the manufacturer's instructions (KeyGEN BioTECH, KGA7072).

[0072] Spermocyte nuclear spreading and immunofluorescence staining

[0073] In order to obtain a reliable single-cell suspension of testicular cells, the mouse spermatocyte meiotic chromosome spreading method was optimized based on previous studies (Dia, F., Strange, T., Liang, J., Hamilton, J., and Berkowitz, KM (2017). Preparation of Meiotic Chromosome Spreads from Mouse Spermatocytes. Journal of visualized experiments: JoVE.). The testicles of male mice were removed and the tunica albuginea was removed. The seminiferous tubules were treated in a hypotonic buffer (30mM Tris, 50mM sucrose, 17mM sodium citrate trisodium, 5mM EDTA, 2.5mM dithiothreitol and 1mM phenylmethylsulfonyl fluoride, pH 8.2) for 40 minutes. Subsequently, the seminiferous tubules were gently squeezed in 100mM sucrose buffer (pH 8.2). The squeezed tubules were resuspended in 100mM sucrose solution and gently spread on a pretreated slide. The slides were treated with 1% paraformaldehyde buffer (pH 9.2, adjusted with 10 mM boric acid) containing 0.15% Triton X-100. Then, the slides were incubated in a humidified chamber at room temperature for at least 3 hours, air-dried, washed twice with 0.4% optical flow agent solution, and finally air-dried at room temperature and stored in a -80°C refrigerator until use.

[0074] For immunofluorescence staining, slides were blocked with 5% BSA for 1 h at room temperature and then incubated with primary antibodies (see Table S2) overnight at 4°C. After washing, slides were incubated with secondary antibodies and washed three times with PBS before mounting. The primary antibodies used were as follows: mouse anti-SYCP3 (1:200 dilution; Abcam, ab97672), rabbit anti-SYCP1 (C-terminal) (1:200 dilution; Abcam, ab15090), phospho-histone H2A.X (Ser139) (20E3) rabbit monoclonal antibody (1:500 dilution; Cell Signaling Technology, 9718S). Laser scanning confocal images were acquired using a confocal microscope (LSM710), and fluorescence signals were analyzed using ImageJ and ZEN software.

[0075] RNA-seq

[0076] Testicular cells from wild-type and DDX17 testis-specific knockout mice were isolated. Cells were lysed using TRIzol reagent, and after chloroform separation of the supernatant, RNA was precipitated with isopropanol. The RNA precipitate was washed with 75% ethanol and dissolved in diethylpyrocarbonate-treated water. The concentration and purity of RNA were assessed using NanoDrop and Qubit, and its integrity was confirmed using an Agilent Bioanalyzer (RIN ≥ 7.0). To remove genomic DNA contamination, RNA was treated with DNase I and purified using an RNA purification kit. cDNA libraries were constructed from 1 μg of high-quality RNA using the NEB Next Ultra RNA Library Preparation Kit. mRNA was enriched by polyadenylation capture, reverse transcribed into double-stranded cDNA, and adapter construction was performed by end repair and A-tailing. Library quality was assessed and quantified using Qubit and Bioanalyzer 2100 before loading the library into the Illumina NovaSeq 6000 platform for high-throughput sequencing (PE150), ensuring at least 20 million valid reads per sample. Raw sequencing data were quality controlled using FastQC, and low-quality reads and adapter sequences were trimmed using Trimmomatic software. Clean data were aligned to the mouse reference genome (mm10) using HISAT2, and transcript quantification was performed using FeatureCounts. Differential expression analysis was performed using DESeq2 to identify significantly differentially expressed genes, with a threshold of |log2FC|≥1 and a corrected p-value <0.05. Selected differentially expressed genes were further validated by qRT-PCR.

[0077] Micro RNA-Seq

[0078] The library was prepared using MGI's TruePrep DNA library preparation kit (VAZYME, China). RNA input was less than 4 μL, used as a template, and amplified using Discover-sc WTA Kit V2 (VAZYME, China). Reverse transcription was performed using oligodT primers to synthesize cDNA, and then full-length cDNA was amplified by SMART-SeqII reaction. The resulting amplified cDNA was treated using a transposon-based method to construct the final library. Subsequently, single-stranded library products were generated by denaturation. Circularization was performed to produce single-stranded circular DNA molecules, and linear DNA molecules were digested. The circular library was then amplified using phi29 polymerase and rolling circle amplification (RCA) to create DNA nanoballs (DNBs) carrying more than 300 copies of the initial circular DNA molecules. These DNBs were loaded onto the patterned nanoarray and paired-end reads of 100 / 150 bases in length were generated on the G400 / T7 / T10 sequencing platform (BGI-Shenzhen, China).

[0079] Integrative analysis of mouse testis single-cell RNA-seq data and bulk RNA-seq data

[0080] The gene expression matrix data of mouse testicular cells were obtained from the GEO database, which was provided by Chen et al. (Chen, Y., Zheng, Y., Gao, Y., Lin, Z., Yang, S., Wang, T., Wang, Q., Xie, N., Hua, R., Liu, M., et al. (2018).). This dataset was generated by SMART-seq2 technology, covering 20 developmental stages during synchronous spermatogenesis, and contained a total of 1,204 single cells, covering mitosis, meiosis, and late meiotic stages. After strict quality control, 1,167 single-cell sequencing datasets were retained for subsequent analysis. According to the data processing pipeline described in detail in Chen's study, the Seurat software package was used for preliminary analysis. The quality control criteria included: the number of genes detected per cell exceeded 2,000, and the unique molecular identifier (UMI) count was between 20,000 and 1,000,000. Gene expression levels were normalized using log2(TPM / 10+1), and 15 principal component dimensions (PC1–PC15) were selected for t-SNE analysis. DotPlot was used to visualize the expression of cell markers.

[0081] The 24-hour follow-up was performed according to a previously published protocol (Hermann, BP, Cheng, K., Singh, A., Roa-De LaCruz, L., Mutoji, KN, Chen, I.C., Gildersleeve, H., Lehle, JD, Mayo, M., B., et al. (2018). The Mammalian Spermatogenesis Single-Cell Transcriptome, from Spermatogonial Stem Cells to Spermatids. Cell reports 25, 1650-1667.e1658.; Lukassen, S., Bosch, E., Ekici, AB, and Winterpacht, A. (2018). Single-cell RNA sequencing ofadultmouse tests. Sci Data 5, 180192.; Zhang, J., Lu, T., Lu, S., Ma, S., Han, D., Zhang, K., Xu, C., Liu, S., Gan, L., Wu, X., et al. (2023). Single-cell analysis of multiple cancer types reveals differences in endothelial cells between tumors and normal tissues. Computational andstructuralbiotechnologyjournal 21,665-676.), the AggregateExpression function was used to convert the single-cell RNA-seq data into pseudo-bulk RNA-seq gene expression matrices (count values) of different cell types. The testicular cell bulk RNA-seq data obtained by R&DFACS and the pseudo-bulk RNA-seq matrix from the single-cell transcriptome were normalized using log2(CPM+1). Finally, the corrplot package was used for correlation analysis to classify the testicular cell types in bulk RNA-seq based on gene expression data.

[0082] Test results:

[0083] To further elucidate the identities of these cell populations, a detailed comparative analysis of the nine cell populations and their classical Hoechst fluorescence signatures was performed. It was found that the nine groups of cells could be identified by developmental trajectories marked by classical Hoechst dyes (via Hoechst red and Hoechst blue) rather than being scattered throughout the developmental trajectories, suggesting that the cell populations distinguished by R&D-FACS are subtypes of cells at specific stages of spermatogenesis ( Figure 3 A).

[0084] To accurately determine the identities of these nine cell populations, R&D-FACS sorting was performed, followed by low-input RNA sequencing to analyze the specific gene expression profiles of each group of spermatogenic cells ( Figure 3 B in Figure 2 shows the workflow of low-input RNA sequencing. In addition, the SMART-seq2 single-cell transcriptome gene expression matrix provided by Chen et al., which covers various stages of spermatogenesis in mouse testicular cells, was used (Chen, Y., Zheng, Y., Gao, Y., Lin, Z., Yang, S., Wang, T., Wang, Q., Xie, N., Hua, R., Liu, M., et al. (2018). Single-cell RNA-seq uncovers dynamic processes and critical regulators in mouse spermatogenesis. Cell Res 28, 879-896.). Based on these data, a pseudo-bulk RNA-seq dataset was generated. Following the data processing methods described in the original study, t-SNE analysis was performed, and the DotPlot function was applied to visualize the expression of cell-specific marker genes. Finally, the low-input RNA-seq results of these nine cell populations were cross-referenced with the pseudo-bulk RNA-seq dataset. Combined with the positions of these populations in the classic Hoechst fluorescence characteristic map (Gaysinskaya, V., Soh, IY, van der Heijden, GW, and Bortvin, A. (2014). Optimized flow cytometry isolation of murine spermatocytes. Cytometry A 85, 556-565.) and identification by chromosome spreading and immunofluorescence staining, the types of these nine cell populations were finally determined.

[0085] In the R&D-FACS characteristic plot, P1 exhibits diploid DNA content and is located at a position corresponding to spermatogonia in the classical fluorescence plot ( Figure 3 A in Figure 2). Low-input RNA-seq analysis revealed that P1 highly expressed markers of differentiated spermatogonia, including Stra8, Uchl1, Crabp1, and Rhox13. Furthermore, Pearson correlation analysis with the pseudo-bulk RNA-seq dataset showed that the transcriptional profile of P1 was highly similar to that of differentiated spermatogonia (A1, In phase, S phase B-type, and G2 / M phase B-type spermatogonia) and pre-leptotene spermatocytes. Therefore, it was determined that the P1 population represented differentiated spermatogonia ( Figure 3 C).

[0086] P2 exhibits tetraploid DNA content and is localized at the L / Z position in the Hoechst fluorescence signature ( Figure 3 Figure 2 (A). Low-input RNA-seq analysis revealed that P2 highly expressed markers of leptotene and zygotene spermatocytes, such as Gm960, Meiob, and Dmc1. Pearson correlation analysis with the pseudo-bulk RNA-seq dataset showed that the transcriptional profile of P2 was highly similar to that of leptotene spermatocytes (sc-L), zygotene spermatocytes (sc-Z), differentiating spermatogonia, and pre-leptotene spermatocytes. Therefore, the P2 population was mainly composed of leptotene and zygotene spermatocytes.

[0087] P3 and P4 are both tetraploid cells and are located at the P / D position in the Hoechst fluorescence characteristic map ( Figure 3 A in Figure 1). Low-input RNA-seq results showed that both P3 and P4 highly expressed Psma8, Piwil1, and Pou5f2, indicating that they correspond to pachytene / diplotene spermatocytes ( Figure 3 C in Figure 2). The expression of Ccna1 in P4 was significantly higher than that in P3, suggesting that P4 may represent the diplotene stage, while P3 corresponds to the pachytene stage. Principal component analysis (PCA) and Pearson correlation analysis revealed a strong correlation between P3 and P4 ( Figure 3 D in ). By comparing the low-input RNA-seq results with pseudo-bulk RNA-seq data, it was found that P3 and P4 were closely related to the P and D populations. Chromosome spreading and immunofluorescence staining of P3 and P4 sorted by R&D-FACS confirmed that P3 was mainly composed of pachytene spermatocytes, while P4 was mainly composed of diplotene spermatocytes, as indicated by SYCP3 and H2AX staining ( Figure 4 Thus, the P3 population corresponds to pachytene spermatocytes, whereas P4 represents diplotene spermatocytes.

[0088] Comprehensive analysis of the R&D-FACS fluorescence profiles showed that the DNA content of P5 was between 2C and 4C ( Figure 3 This population highly expressed the diplotene spermatocyte markers Psma8 and Piwil1, as well as elevated classical meiotic division II markers Ccna1 and Tbp1 ( Figure 3 C in Figure 3). PCA and Pearson correlation analysis of the low-input RNA data showed a strong correlation among P5, P6, and P7 ( Figure 3 In addition, the mRNA expression profile of P5 was highly similar to that of the sc-MII and sc-D populations in pseudo-bulk RNA-seq data ( Figure 3 Based on the above analysis, the P5 population corresponds to cells in the late stage of the first meiotic division (MI).

[0089] Both P6 and P7 showed diploid DNA content and were localized at the MII position in the Hoechst fluorescence profile ( Figure 3 Low-input RNA-seq analysis showed that the mRNA expression profiles of the two populations were highly similar, and both subpopulations highly expressed the meiotic division II (MII) marker genes Ccna1 and Tbp1 ( Figure 3 C). Correlation analysis with the pseudo-bulk RNA-seq dataset confirmed that the mRNA profiles of these two populations were highly similar to those of the spermatid remnant body (sc-RS) and meiotic second stage spermatid (sc-MII) populations ( Figure 3 E). Therefore, the P6 and P7 populations represent two subpopulations of secondary spermatocytes (MII).

[0090] P8 and P9 are both haploid cells, indicating that they have undergone the second meiotic division and developed into gametes ( Figure 3 In addition, low-input RNA-seq results showed that P8 highly expressed round spermatid markers, such as Sun5, Tex29, and Spaca3, while P9 was characterized by highly expressed elongated spermatid markers Prm1, Prm2, Ttc24, Reep6, and Cstl1 ( Figure 3 DAPI staining of P8 and P9 populations sorted by R&D-FACS confirmed that P8 was mainly composed of round spermatids, while P9 was mainly composed of elongated spermatids ( Figure 4 B).

[0091] In summary, the R&D-FACS provided by the present invention not only expands the capabilities of traditional Hoechst-based FACS, realizes the separation of known cell populations, but also can accurately classify cell types that were previously difficult to distinguish, such as pachytene spermatocytes, diplotene spermatocytes, round spermatocytes, and elongated spermatocytes. The method also successfully distinguishes two MII cell subtypes and identifies cells in the first meiotic division (MI) M phase. These experimental results demonstrate that the R&D-FACS method provided by the present invention has excellent accuracy and versatility in characterizing spermatogenic cell populations.

[0092] Example 3

[0093] Combination of R&D-FACS and capillary electrophoresis reveals unique RNA signatures of P and D spermatocytes and MII and round spermatocytes

[0094] RNA fingerprinting by capillary electrophoresis

[0095] Mouse testicular cells were obtained as described previously and expressed using Hoechst 33342 / RNASelectTM Green fluorescent cell stain / propidium iodide (PI) staining was used to obtain 9 groups of cells by flow cytometry (FACS). Total RNA was extracted using ENZATotal RNAkit I. RNA was denatured at 70°C for 5 minutes in a thermal cycler, then quickly placed on ice for 5 minutes to stabilize, and analyzed by capillary electrophoresis using an automated nucleic acid / protein analysis system (BIOPTIC, Qsep1).

[0096] The above Example 2 has fully characterized the cell populations identified by R&D-FACS. In further studies, R&D-FACS sorting was combined with capillary electrophoresis to successfully map the RNA fingerprints of these cell populations and generate RNA distribution heat maps for each group ( Figure 4 C in ).

[0097] Capillary electrophoresis results showed that the abundance and ratio of rRNA and mRNA in different cell populations were significantly different ( Figure 4 Although the mRNA transcription level of diplotene spermatocytes (D) was higher than that of pachytene spermatocytes (P), R&D-FACS analysis showed that the total RNA abundance of P was lower than that of D ( Figure 2 D in the figure). Analysis of RNA fingerprints showed that the rRNA level in pachytene spermatocytes was significantly higher than that in diplotene spermatocytes ( Figure 4 This suggests that, despite the higher transcriptional activity of D cells, they undergo greater rRNA depletion, resulting in reduced total RNA abundance compared to P.

[0098] The R&D-FACS provided by the present invention can distinguish the second meiotic sperm cells (MII) into two subgroups according to RNA abundance: P6 (MII1) and P7 (MII2) ( Figure 3 Further analysis of RNA fingerprints showed that the RNA fingerprint of MII2 was highly similar to that of P8 (round spermatids, RS) ( Figure 4 E), indicating a close developmental relationship between the two. Therefore, it can be concluded that in MII2 cells, the RNA type has already changed to that of round spermatids before undergoing MII division.

[0099] These findings not only deepen our understanding of the dynamic changes of RNA during sperm cell development, but also further verify the powerful ability of R&D-FACS combined with capillary electrophoresis to accurately analyze the RNA characteristics of cell populations. The R&D-FACS method provides new perspectives and tools for the study of germ cell development and helps to reveal the complex molecular mechanisms of sperm production.

[0100] Example 5

[0101] Evaluation of RNA binding protein function using R&D-FACS of the present invention

[0102] animal

[0103] Using CRISPR / Cas9 technology, loxP sites were inserted upstream and downstream of the second exon of the DDX17 gene to generate DDX17-flox mice. Also using CRISPR / Cas9 technology, a 2A-EGFPCre box was knocked into the site before the stop codon of the Stra8 gene to construct Stra8-Cre mice. This design mimics the endogenous expression pattern of the Stra8 gene and ensures that the inserted element does not interfere with the normal function of the Stra8 gene. DDX17-flox mice were crossed with Stra8-Cre mice to generate testis-specific DDX17 knockout mice (abbreviated as DDX17cko). All transgenic strains were maintained on a C57BL / 6J genetic background. The mouse model was maintained in a specific pathogen-free environment at the Animal Center of the Air Force Medical University. All experimental procedures were approved by the Institutional Animal Care and Use Committee (approval number: #20241079) and strictly followed the guidelines for the care and use of experimental animals established by the university.

[0104] Although DDX17cKO mice appeared healthy overall, male mice were infertile. Autopsies revealed that the testicles of adult DDX17cKO male mice were significantly smaller and their testicular weight was significantly reduced ( Figure 5 A in the figure). Fluorescence staining of testicular sections of control mice showed that DDX17 was strongly expressed in the seminiferous tubules within spermatogonia, while no DDX17 signal was found in the lumen of the seminiferous tubules of the testes of DDX17cKO mice, confirming the validity of the knockout model ( Figure 5 D in Figure 1). Histological analysis further revealed that there were large cavities in the seminiferous tubules of DDX17cKO mice, severe loss of spermatogonia, and almost no mature sperm in the epididymis ( Figure 5 TUNEL staining showed that apoptosis was significantly increased in the testis of DDX17cKO mice ( Figure 5 C). These findings suggest that loss of DDX17 disrupts spermatogenesis and ultimately leads to male infertility.

[0105] Interestingly, analysis of chromosome spreading of spermatogonia in DDX17cKO mice (for specific operations, see Example 2) showed that meiotic progression was not completely stagnant, as spermatogonia at different stages of development were still present. In addition, the classical Hoechst fluorescence characteristics of the testes of DDX17cKO mice did not show a significant loss of spermatogonia. One possible reason is that Stra8-mediated Cre recombinase activity does not occur in all spermatogonia, which makes it difficult to study the function of DDX17. In order to further study the potential mechanism of infertility in DDX17cKO mice, R&D-FACS analysis was performed, and the results showed that the RNA abundance of spermatogonia in DDX17cKO mice increased significantly when they entered the DNA replication stage and developed into pre-elongated spermatocytes (pre-L). However, as these cells progressed to the leptotene and zygotene (L / Z) stages, RNA levels gradually returned to control group levels ( Figure 5 E). These findings indicate that knockdown of DDX17 leads to an aberrant increase in RNA expression in pre-leptotene cells at the onset of meiosis.

[0106] Given that R&D-FACS localized the spermatogenesis defect of DDX17cKO mice to the preleptotene stage, we next examined key meiotic events, such as the formation and repair of DNA double-strand breaks (DSBs) and chromosome synapsis. Immunofluorescence staining of SYCP3 and γH2AX showed that in DDX17cKO pachytene spermatocytes, strong γH2AX signals appeared not only around the sex chromosomes but also scattered on the autosomes, indicating a defect in DSB repair ( Figure 5 In addition, co-staining of SYCP1 and SYCP3, which mark the central and axial elements of the synaptonemal complex, showed that the SYCP1 signal was significantly reduced in DDX17cKO pachytene spermatocytes, indicating abnormal synapsis of homologous chromosomes ( Figure 5 G in.

[0107] To evaluate the effect of DDX17 knockout on transcription, R&D-FACS was used to separate pre-elongated spermatocytes and L / Z spermatocytes of DDX17cKO mice, and transcriptome sequencing was performed, which revealed that after DDX17 knockout, gene expression in the pre-elongated spermatocyte and L / Z spermatocyte stages was widely dysregulated. In the L / Z stage, 3336 genes were downregulated and 1283 genes were upregulated, while in the pre-elongated spermatocyte stage, 1668 genes were downregulated and 685 genes were upregulated ( Figure 6 GO analysis of the down-regulated genes revealed that processes related to reproduction, gamete formation, and spermatogenesis were significantly enriched ( Figure 6Further analysis of key genes involved in critical meiotic events, such as DSB repair, recombination, and synapsis, revealed significant differences in gene expression between the two spermatocyte stages ( Figure 6 C).

[0108] DDX17 is a splicing regulator that plays a key role in RNA metabolism. We analyzed alternative splicing (AS) events using the rMATS algorithm and found that 6749 AS events were significantly altered in pre-elongated spermatocytes and 5866 AS events were significantly altered in L / Z spermatocytes after DDX17 knockout. Many key genes involved in synapsis, meiotic recombination, and DSB repair showed significant changes in AS events in both cell populations ( Figure 6 D).

[0109] Our results suggest that DDX17 plays a key role in regulating transcriptional and post-transcriptional events during spermatogenesis. Loss of DDX17 disrupts RNA transcription and alternative splicing, leading to defects in DSB repair and homologous chromosome synapsis, ultimately resulting in spermatogenesis failure and male infertility.

[0110] It can be seen from the above embodiments that the present invention can more accurately distinguish cell types that were previously difficult to distinguish by double staining RNA and DNA in cells; the R&D-FACS provided by the present invention provides an effective means for further detailed research on the spermatogenesis process and provides an effective method for the study of sperm abnormalities and infertility.

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. RNA and DNA dual fluorescence activated cell sorting, characterized in that, The following steps are involved: 1) Digest the cell tissue to obtain a single cell suspension; 2) Double staining of DNA and RNA in the digested single cell suspension; 3) Perform flow cytometry analysis and fluorescence activated cell sorting on the stained cells; The DNA staining solution is Hoechst 33342, and the RNA staining solution is RNASelect TM Green fluorescent cell stain.

2. The RNA and DNA dual fluorescence activated cell sorting method according to claim 1, characterized in that: Step 2) The double staining is to mix the DNA staining solution Hoechst 33342 and the RNA staining solution RNASelect TM Green fluorescent cell stain and single cell suspension were mixed and incubated.

3. The RNA and DNA dual fluorescence activated cell sorting method according to claim 2, characterized in that: The incubation temperature is 36-38°C, and the incubation time is 25-35 minutes; After the cells were digested into single cells, they were resuspended in PBS solution to obtain a single cell suspension. The cell concentration in the single cell suspension was 1 to 9×10 5 / mL.

4. The RNA and DNA dual fluorescence activated cell sorting method according to claim 2, characterized in that: The dosage of the DNA staining solution Hoechst 33342 is 3 to 8 μg / 10 6 cells; the RNA staining solution RNASelect TM The dosage of green fluorescent cell dye is 450~550nmol / 10 6 cells.

5. Use of the RNA and DNA dual fluorescence activated cell sorting method according to any one of claims 1 to 4 in cell subtyping.

6. The use according to claim 5, characterized in that: The cell subtyping includes spermatogenic cell subtyping.

7. The use according to claim 5, characterized in that: The spermatogenic cell subtyping classifies testicular single cells into the following groups: differentiated spermatogonia, Pre-Leptotene spermatocytes, Leptotene / Zygotene spermatocytes, Pachytene spermatocytes, Diplotene spermatocytes, MI spermatocytes, MII spermatocytes, round spermatids, elongated spermatids and Sertoli supporting cells.

8. Use of the RNA and DNA dual fluorescence activated cell sorting method according to any one of claims 1 to 4 in the study of cell genetic material.

9. The use according to claim 8, characterized in that: The genetic material studied includes changes in RNA abundance.

10. Use of the RNA and DNA dual fluorescence activated cell sorting method according to any one of claims 1 to 4 in functional studies of RNA binding proteins.

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