Preparation method of insect wing bud single-cell suspension

Abstract

The invention discloses an insect wing bud single-cell suspension preparation method, which specifically comprises: dissecting in a PBS solution to obtain complete wing bud tissues of five-year-old hemiptera insects, placing in a low-calcium-magnesium PBS solution, centrifuging, carrying out enzymolysis until no cell mass exists to obtain a wing bud tissue mixture, filtering through a 30 [mu] m cell sieve, adding the low-calcium-magnesium PBS solution to terminate enzymolysis, centrifuging, and drying to obtain the insect wing bud single-cell suspension. Collecting a cell precipitate; and cleaning the cell precipitate by using a low-calcium-magnesium PBS solution, centrifuging again to obtain the precipitate, and re-suspending again to obtain the wing bud single-cell suspension. The prepared low-calcium-magnesium PBS solution is used for separating fin bud tissue cells of insect membranous wings, long-time cell activity can be maintained, the concentration of the prepared calcium and magnesium ions is compatible with that of a single-cell sequencing reagent, and the low-calcium-magnesium PBS solution can be directly used for single-cell sequencing.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology and relates to a method for preparing an insect wing bud single cell suspension. Background Art

[0002] Wing polymorphism in insects is one of the most significant features of phenotypic plasticity, and this phenomenon is common in insects. Wing polymorphism is one of the strategies for insects to adapt to external environmental conditions, and it is also the result of a trade-off between flight ability and fertility. Understanding the differences in the developmental patterns of insect wings will help reveal the molecular mechanism of insect wing polymorphism and its ecological significance in the process of biological evolution, and has important theoretical and practical significance for the biological control of agricultural and forestry pests. At present, the research on insect wing types is mainly focused on the molecular and genetic levels, and there is a lack of analysis of the molecular mechanism and evolutionary process of insect wing development at the cell type level.

[0003] Brown planthopper (Nilaparvata lugens (Stal)) belongs to the order Hemiptera, family Delphacidae, and is an important rice pest in Asia; Pyrrhocoris apterus belongs to the order Hemiptera, family Pyrrhocoris. Both have typical wing dimorphism and are excellent models for studying the developmental patterns of insect wings. Insect wings are composed of millions of highly differentiated and interconnected cells. To explore the key cell types that regulate wing development, single-cell sequencing technology is needed. Obtaining a single-cell suspension of insect wing bud tissue is a prerequisite for single-cell sequencing of insect wing buds. However, the cell washing solutions and cell culture media commonly used in single-cell sequencing for mammals and insects are not suitable for insect wing buds, especially insect membranous wings, for the following reasons: Insect wing bud cells are fragile and sensitive to pH and osmotic pressure. Commonly used cell washing solutions and cell culture media have low salt ion concentrations and osmotic pressures, which cause cells to absorb water and swell, thereby destroying the integrity of cells and failing to achieve the high cell survival rate required for single-cell sequencing. In addition, in order to ensure the success and accuracy of single-cell sequencing, a buffer without calcium and magnesium ions is usually used to prepare a single-cell suspension, because calcium and magnesium ions may affect the reaction of the reverse transcription system in oil-in-water, thereby causing reverse transcription failure. However, the cell survival rate is low after the insect wing bud cells are treated with the buffer. In view of the above problems, it is urgent to study a suitable reagent that meets the requirements of single-cell sequencing while targeting the characteristics of insect wing bud cells and meeting high cell survival rates. The present invention achieves efficient dissociation of insect wing buds in a relatively short time by using trypsin-EDTA solution and a homemade low-calcium and magnesium PBS solution, while taking into account cell survival rate, and the prepared wing bud cell suspension meets the sequencing requirements of the 10×genomics platform. Summary of the invention

[0004] The object of the present invention is to provide a method for preparing an insect wing bud single cell suspension in view of the above problems.

[0005] In order to achieve the above object, the present invention adopts the following technical scheme: As a first aspect, the present invention provides a method for preparing a single cell suspension of insect wing buds, the method comprising the following steps: Take 5th instar Hemiptera insects, dissect them in 1 mL phosphate buffered saline (PBS solution) to obtain complete wing bud tissue, place them in 1 mL low calcium and magnesium PBS solution, and centrifuge; Add 1 mL of protease solution to the centrifuged wing bud tissue and perform enzymatic hydrolysis at 37°C until there is no cell mass, thereby obtaining a wing bud tissue mixture; After the wing bud tissue mixture was filtered through a 30 μm cell sieve, a low calcium and magnesium PBS solution was added to terminate the enzymatic hydrolysis, so that the total volume of the filtered wing bud tissue mixture and the low calcium and magnesium PBS solution was 10 mL, and the mixture was centrifuged to collect the cell precipitate; the cell precipitate was washed with a low calcium and magnesium PBS solution, centrifuged to obtain a precipitate, and 1 mL of the low calcium and magnesium PBS solution was added to resuspend the precipitate again to obtain a wing bud single cell suspension; The low calcium and magnesium PBS solution is prepared by the following steps: CaCl2 and MgSO4 are added to the PBS solution so that the final concentration of CaCl2 is 500 mg / L, the final concentration of MgSO4 is 920 mg / L, the osmotic pressure is 360 mOsm / kg, and the pH value is adjusted to 6.2 using sodium hydroxide (NaOH) or hydrochloric acid (HCl).

[0006] Furthermore, the protease solution is a trypsin-EDTA solution containing 2.5 g / L trypsin and 0.2 g / L EDTA, and is preheated at 37°C before being added to the wing bud tissue.

[0007] Preferably, the wing bud tissue is centrifuged in a 1.5 mL Eppendorf tube at 4°C and 500 RCF (relative centrifugal force) for 5 minutes.

[0008] Preferably, the enzymatic hydrolysis conditions of the wing bud tissue are 37° C. metal bath, enzymatic digestion for 5 to 7 minutes, and blowing the cell clumps 1 to 2 times with a pipette.

[0009] Furthermore, the Hemiptera insect is brown planthopper or red bug.

[0010] Preferably, the filtered wing bud tissue mixture and the cell pellet washed with a low calcium and magnesium PBS solution are centrifuged in a 15 mL centrifuge tube at 4° C. and 300 RCF for 5 min.

[0011] As a second aspect, the present invention provides an insect wing bud single cell suspension prepared by the above preparation method.

[0012] As a third aspect, the present invention provides an application of the above-mentioned insect wing bud single-cell suspension in single-cell sequencing.

[0013] The present invention has the following beneficial effects: the present invention provides a method for preparing a cell suspension, which is suitable for insect wing bud tissue, and in particular, uses the low calcium and magnesium PBS solution created by the present invention to separate wing bud tissue cells of insect membranous wings, and the separated single cells have high purity, can maintain cell activity for a long time, have no agglomeration phenomenon, have few impurities and fragments, and have a clean background. The separated cell suspension can be directly used for single-cell sequencing. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 Bright field and fluorescence field images of a single cell suspension of a brown planthopper wing bud for single cell sequencing provided in an embodiment of the present invention under a Luna-fl automatic fluorescence cell counter, wherein (a) is a bright field image and (b) is a fluorescence field image; Figure 2 Bright field and fluorescence field images of a single cell suspension of the wing bud of a red bug for single cell sequencing provided in an embodiment of the present invention under a Luna-fl automatic fluorescence cell counter, wherein (a) is a bright field image, and (b) is a fluorescence field image; Figure 3 A curve chart showing the number of captured cells and the number of UMIs of a single cell suspension of a brown planthopper wing bud provided in an embodiment of the present invention; Figure 4 A curve chart showing the number of captured cells and the number of UMIs of a single-cell suspension of the wing bud of a red bug provided in an embodiment of the present invention. DETAILED DESCRIPTION

[0015] The following is a description of the implementation of the present invention by specific embodiments. People familiar with the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0016] In the present invention, the PBS solution was purchased from ThermoFisher, USA (Cat. No. 10010023), and the pH value was 7.4. The low calcium and magnesium PBS solution was prepared in the present invention: CaCl2 and MgSO4 were added to the PBS solution, so that the final concentration of CaCl2 was 500 mg / L, the final concentration of MgSO4 was 920 mg / L, the osmotic pressure was 360 mOsm / kg, and NaOH or HCl was added to adjust the pH value to 6.2; the trypsin-EDTA solution was purchased from Sigma, USA (Cat. No. T4049).

[0017] Example 1: Method for preparing single cell suspension of brown planthopper wing buds.

[0018] The method for preparing the brown planthopper wing bud single cell suspension of the present embodiment comprises the following steps: Take 50 5th-instar (48-54h) brown planthoppers, separate the complete brown planthopper wing bud tissue in PBS solution (1 mL in this example) with dissecting forceps, and wash it with PBS for 3-4 times. Transfer the washed tissue to a 1.5 mL Eppendorf tube containing 1 mL low-calcium and magnesium PBS solution (pH=6.2), and centrifuge it at 4°C and 500RCF for 5 minutes.

[0019] Add 1 mL of 37°C preheated trypsin-EDTA solution (containing 2.5 g / L trypsin and 0.2 g / L EDTA) to the wing bud tissue, place it in a 37°C metal bath for full digestion for 5-7 min, and gently blow the cell clumps 1-2 times with a pipette to fully dissociate them until there are no cell clumps, thus obtaining a mixture of brown planthopper wing bud tissue.

[0020] The wing bud tissue mixture was filtered into a 15 mL centrifuge tube using a 30 μm cell sieve, supplemented with low calcium and magnesium PBS solution to 10 mL, and centrifuged at 4°C and 300 RCF for 5 min. After obtaining the cell precipitate, the precipitate was washed with low calcium and magnesium PBS solution, centrifuged again under the same conditions, and after obtaining the precipitate, 1 mL of low calcium and magnesium PBS solution was added and resuspended to obtain a single cell suspension of brown planthopper wing buds.

[0021] Example 2: Method for preparing single cell suspension of wing buds of Trichoderma spp.

[0022] The method for preparing the wing bud single cell suspension of the present embodiment comprises the following steps: Take 20 5-year-old (5d) red bugs, separate the complete wing bud tissue of the red bugs with dissecting forceps in PBS solution (1 mL in this example), and wash it with PBS for 3-4 times. Transfer the washed tissue to a 1.5 mL Eppendorf tube containing 1 mL low calcium and magnesium PBS solution (pH=6.2), and centrifuge it at 4°C and 500RCF for 5 minutes.

[0023] Add 1 mL of 37°C preheated trypsin-EDTA solution (containing 2.5 g / L trypsin and 0.2 g / L EDTA) to the wing bud tissue, place it in a 37°C metal bath for full digestion for 5-7 min, and gently blow the cell clumps 1-2 times with a pipette to fully dissociate them until there are no cell clumps, thus obtaining a mixture of wing bud tissue of the red bug.

[0024] The wing bud tissue mixture was filtered into a 15 mL centrifuge tube using a 30 μm cell sieve, supplemented with low calcium and magnesium PBS solution to 10 mL, and centrifuged at 4°C and 300 RCF for 5 min. After obtaining the cell precipitate, the precipitate was washed with low calcium and magnesium PBS solution, centrifuged again under the same conditions, and after obtaining the precipitate, 1 mL of low calcium and magnesium PBS solution was added and resuspended to obtain a single cell suspension of the wing bud of the red bug.

[0025] Example 3: Preparation of insect wing bud single cell suspension using common cell washing solution and cell culture medium.

[0026] The common cell washing solution and cell culture medium for preparing insect wing bud single cell suspension in this embodiment include the following: Single cell suspensions of wing buds of R. lugens and N. lugens were prepared using PBS solution with a pH of 7.4 and an osmotic pressure of 300 mOsm / kg, excluding calcium and magnesium ions.

[0027] Schneider insect medium, purchased from Merck, Germany (Cat. No. S0146), with a pH of 6.4 and an osmotic pressure of 340 mOsm / kg, without calcium and magnesium ions, was used to prepare single-cell suspensions of the wing buds of R. elegans and N. lugens.

[0028] DMEM high-glucose medium, purchased from Silarbio, China (Cat. No. 11995), with unknown pH and osmotic pressure, was used to prepare single-cell suspensions of the wing buds of R. elegans and N. lugens.

[0029] Grace's supplemented insect cell culture medium was purchased from Pricella, China (Cat. No. PM15202), pH 6.2, CaCl2 final concentration 500 mg / L, Mg 2+ The final concentration was 3638 mg / L, and single cell suspensions of wing buds of R. lugens and N. lugens were prepared.

[0030] Insect culture medium was used, purchased from Coolaber, China (Cat. No. CM0021), with a final concentration of CaCl2 of 0.6 mg / L, Mg 2+ The final concentration was 1.8 mg / L, and single cell suspensions of wing buds of R. lugens and N. lugens were prepared.

[0031] Test Example 1: The single cell suspension of brown planthopper wing buds prepared in Example 1 was placed at room temperature for 30 minutes, 2 uL was taken and added to 18 uL of AO / PI (acridine orange / propidium iodide) and Trypan blue mixed dyes, and 10 uL was taken and placed in the counting chamber of the counting plate. The bright field and fluorescence field counting results were as follows: Figure 1 As shown, green fluorescence marks live cells and red fluorescence marks dead cells. Figure 1(a) is a bright field image, showing a clean background with no obvious cell debris. Figure 1 (b) is a fluorescence field image, which shows that low calcium and magnesium PBS solution can maintain cell activity for a long time.

[0032] Test Example 2: The single cell suspension of the wing bud of the red bug prepared in Example 2 was placed at room temperature for 30 minutes, 2 uL was added to 18 uL of AO / PI and Trypan blue mixed dye, and 10 uL was placed in the counting chamber of the counting plate after mixing. The bright field and fluorescence field counting results were as follows: Figure 2 As shown, green fluorescence marks live cells and red fluorescence marks dead cells. Figure 2 (a) is a bright field image, showing a clean background with no obvious cell debris. Figure 2 (b) is a fluorescence field image, which shows that low calcium and magnesium PBS solution can maintain cell activity for a long time.

[0033] The single cell suspensions of Example 1 and Example 2 were counted using a Luna-fl automatic fluorescent cell counter to obtain a cell counting report. The results are shown in Table 1.

[0034] Table 1 Comparison of single cell suspensions of wing buds of brown planthopper and red bug

[0035] As can be seen from Table 1, the number of single cells obtained by the method for preparing insect wing bud single-cell suspension involved in the present invention meets the requirements of single-cell sequencing, and the cell viability is high, the agglomeration rate is low, and the preparation effect is good.

[0036] Experimental Example 3: The single cell suspension of brown planthopper wing buds obtained in Example 1 was subjected to 10×Genomics single cell transcriptome sequencing, and the operation was performed according to the standard operation procedure on the 10×Genomics official website. The results are as follows: Figure 3 As shown. The total number of sequenced cells of brown planthoppers was 13,040, the average number of reads per cell was 37,205, and the median number of genes per cell was 1,850. The total number of reads of single-cell sequencing was 485,158,812, with valid barcodes accounting for 97.0%, valid UMIs (Unique Molecular Identifiers) accounting for 99.9%, sequencing saturation was 57.9%, Q30 bases in barcodes accounted for 96.0%, Q30 bases in RNA reads accounted for 92.4%, and Q30 bases in UMIs accounted for 94.5%. Reads matched the genome at 80.8%, and the proportion of reads uniquely aligned to the genome was 64.5%. Figure 3The figure is a barcode rank plot of the number of captured cells and the number of UMIs. The proportion of cells matching the genome is 89.2%.

[0037] Experimental Example 4: The single cell suspension of the wing bud of the proto-red bug obtained in Example 2 was subjected to 10×Genomics single cell transcriptome sequencing, and the operation was performed according to the standard operating procedures on the official website of 10×Genomics. The results are as follows: Figure 4 As shown. The total number of sequenced cells of the red bug was 13,637, with an average of 31,101 reads per cell and a median of 1,352 genes per cell. The total number of single-cell sequencing reads was 424,126,854, with valid barcodes accounting for 97.5%, valid UMIs accounting for 100.0%, sequencing saturation of 48.5%, Q30 bases in barcodes accounting for 96.7%, Q30 bases in RNA reads accounting for 94.4%, and Q30 bases in UMIs accounting for 94.9%. Reads matched the genome at 79.5%, and the proportion of reads uniquely aligned to the genome was 77.8%. Figure 4 The figure is a barcode rank plot of the number of captured cells and the number of UMIs. The proportion of cells matching the genome is 59.9%.

[0038] It can be seen from Experimental Examples 3-4 that the present invention uses the low calcium and magnesium PBS solution to prepare a single cell suspension, which meets the sequencing requirements, the sequencing results are of high quality, and the data meet the requirements of downstream single cell data analysis.

[0039] Test Example 5: A series of single cell suspensions of wing buds of brown planthoppers obtained in Example 3 were placed at room temperature for 30 minutes, 2 uL was taken respectively, and each was added to 18 uL of AO / PI and Trypan blue mixed dye. After mixing, 10 uL was taken and placed in the counting chamber of the counting plate, and the cell viability was determined using a Luna-fl automatic fluorescent cell counter. The results are shown in Table 2.

[0040] Table 2 Cell viability of wing bud suspension prepared with cell washing solution and cell culture medium

[0041] As shown in Table 2, wing bud cells are fragile, and commonly used cell washing solutions and cell culture media cannot maintain the activity of wing bud cells and cannot achieve the high cell survival rate required for single-cell sequencing.

[0042] The above shows and describes the basic principles, main features and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. A method for preparing a single cell suspension of insect wing buds, characterized in that: The following steps are involved: Take 5th instar Hemiptera insects, dissect them in 1 mL PBS solution to obtain complete wing bud tissue, place them in 1 mL low calcium and magnesium PBS solution, and centrifuge; Add 1 mL of protease solution to the centrifuged wing bud tissue and perform enzymatic hydrolysis at 37°C until there is no cell mass, thereby obtaining a wing bud tissue mixture; After the wing bud tissue mixture was filtered through a 30 μm cell sieve, a low calcium and magnesium PBS solution was added to terminate the enzymatic hydrolysis, so that the total volume of the filtered wing bud tissue mixture and the low calcium and magnesium PBS solution was 10 mL, and the mixture was centrifuged to collect the cell precipitate; the cell precipitate was washed with a low calcium and magnesium PBS solution, centrifuged to obtain a precipitate, and 1 mL of the low calcium and magnesium PBS solution was added to resuspend the precipitate again to obtain a wing bud single cell suspension; The low calcium and magnesium PBS solution is prepared by the following steps: CaCl2 and MgSO4 are added to the PBS solution so that the final concentration of CaCl2 is 500 mg / L, the final concentration of MgSO4 is 920 mg / L, the osmotic pressure is 360 mOsm / kg, and the pH value is adjusted to 6.2 using HCl or NaOH.

2. The method for preparing the insect wing bud single cell suspension according to claim 1, characterized in that: The protease solution was trypsin-EDTA solution, containing 2.5 g / L trypsin and 0.2 g / L EDTA, and was preheated at 37°C before adding to the wing bud tissue.

3. The method for preparing the insect wing bud single cell suspension according to claim 1, characterized in that: The wing bud tissue is centrifuged in a 1.5 mL Eppendorf tube at 4° C. and 500 RCF for 5 minutes.

4. The method for preparing the insect wing bud single cell suspension according to claim 1, characterized in that: The enzymatic hydrolysis conditions of the wing bud tissue are as follows: the enzymatic hydrolysis is carried out in a metal bath at 37° C. for 5 to 7 minutes, and the cell clumps are blown and beaten with a pipette 1 to 2 times.

5. The method for preparing the insect wing bud single cell suspension according to claim 1, characterized in that: The Hemiptera insect is brown planthopper or red bug.

6. The method for preparing the insect wing bud single cell suspension according to claim 1, characterized in that: The filtered wing bud tissue mixture and the cell pellet washed with a low calcium and magnesium PBS solution were centrifuged in a 15 mL centrifuge tube at 4° C. and 300 RCF for 5 minutes.

7. An insect wing bud single cell suspension prepared by the preparation method according to any one of claims 1 to 6.

8. Use of the insect wing bud single cell suspension according to claim 7 in single cell sequencing.

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