Method for establishing ATAC-seq library by using quinoa microspores

Abstract

The invention provides a method for establishing an ATAC-seq library by using quinoa microspores, and belongs to the technical field of bioengineering. By improving a microspore sample pretreatment process and adjusting parameters of a transposase reaction system, key technical bottlenecks in the prior art are solved, efficient construction of the quinoa microspore ATAC-seq library is realized, and reliable technical support is provided for analyzing a chromatin regulatory network developed by quinoa male gametophytes and excavating and regulating key genes.

Description

Technical Field

[0001] This invention belongs to the field of bioengineering technology, and in particular relates to a method for constructing an ATAC-seq library using quinoa microspores. Background Technology

[0002] Quinoa, a pseudocereal crop with both high nutritional value and strong environmental adaptability, is rich in high-quality protein, unsaturated fatty acids, and various micronutrients, playing a vital strategic role in ensuring food security and improving dietary structure. With the rapid development of molecular breeding technology, elucidating the gene expression regulation mechanisms during quinoa's reproductive development, especially the dynamic changes in chromatin accessibility during the formation of male gametophytes (microspores), has become a key breakthrough for exploring quinoa's superior genetic resources and optimizing breeding strategies.

[0003] ATAC-seq (transposase accessibility chromatin sequencing) technology, as a core tool for studying open chromatin regions, has advantages such as low sample requirements, short experimental cycles, and high resolution, and has been widely used in epigenetic studies of model crops such as Arabidopsis thaliana and rice. However, quinoa microspores have unique cell wall structures, abundant cytoplasmic contents, and cell activity easily affected by the external environment. Existing ATAC-seq library construction methods, when applied to quinoa microspores, suffer from the inability to extract the cell nucleus, resulting in the inability to efficiently obtain high-quality chromatin accessibility data, which severely restricts research on epigenetic mechanisms related to quinoa microspore development. How to achieve efficient construction of quinoa microspore ATAC-seq libraries, providing reliable technical support for elucidating the chromatin regulatory network of quinoa male gametophyte development and identifying key regulatory genes, remains to be further studied. Summary of the Invention

[0004] In view of this, the purpose of this invention is to provide a method for constructing an ATAC-seq library using quinoa microspores.

[0005] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides a method for constructing an ATAC-seq library using quinoa microspores, comprising the following steps: 1) Select unopened quinoa flower buds, extract quinoa microspores, wash with buffer solution to obtain washed microspores; 2) Mix the washed microspores with Tn5 enzyme digestion buffer to obtain a mixture, then terminate the reaction to obtain the enzyme digestion solution; 3) Recover nucleic acids from the enzyme digestion solution using magnetic beads to obtain the ATAC-seq library; The Tn5 enzyme digestion buffer contains the following components at the following concentrations: Sucrose 0.1~0.3 mg / mL, Tris-HCl 0.01~0.1 M, MgCl2 2~6 mM, CaCl2 0.5~2 mM.

[0006] Preferably, the method for extracting quinoa microspores in step 1) includes the following steps: S1. When quinoa begins to flower, take the unflowered quinoa flower buds, treat them at 0~6℃ for 2~6 days, and then sterilize them to obtain sterile flower buds; S2. After extracting the microspores from the flower buds with the extract, centrifuge and discard the supernatant to obtain quinoa microspores; The extract contains the following components at the following concentrations: 40-80 g / L mannitol, 0.5-2 g / L calcium chloride, 0.5-2 g / L MES, and pH 5.0-6.5.

[0007] Preferably, the sterilization in step S1 includes soaking the flower buds in a 5%~20% sodium hypochlorite solution and then washing them 2~6 times; The ratio of quinoa flower buds to sodium hypochlorite solution is 80-12:10-30 mL; The soaking time is 5-20 minutes, and the soaking temperature is 20-30℃.

[0008] Preferably, after washing in step 1), the supernatant is discarded by centrifugation; The cleaning is performed 1 to 5 times; The centrifugation speed is 5000~15000 rpm, and the centrifugation time is 1~3 min.

[0009] Preferably, in step 2), the ratio of the cleaned microspores to the Tn5 enzyme digestion buffer is 3000-7000 microspores: 20-50 μL.

[0010] Preferably, the mixing time in step 2) is 20-40 min, and the mixing temperature is 35-40℃.

[0011] Preferably, step 2) terminates by mixing the digestion buffer solution with the mixture; The mixing temperature is 45~55℃; The mixing time is 20-40 minutes.

[0012] Preferably, step 3) involves recycling with magnetic beads by mixing the enzyme digestion solution with magnetic beads for 3 to 10 minutes.

[0013] Preferably, step 3) after recycling with magnetic beads further includes washing with 60%~100% alcohol.

[0014] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a method for constructing ATAC-seq libraries using quinoa microspores. By improving the microspore sample pretreatment process and adjusting the transposase reaction system parameters, the key technical bottlenecks in the existing technology are solved, and the efficient construction of quinoa microspore ATAC-seq libraries is achieved. This provides reliable technical support for analyzing the chromatin regulatory network of quinoa male gametophyte development and mining key regulatory genes. Attached Figure Description

[0015] Figure 1 Microscopic photograph of quinoa microspores after shaking and disruption; Figure 2 The DNA enriched by enzyme digestion in Example 2 is shown in the electron gel image detected by 4150 TapeStation. Figure 3 The image shows an electronic gel image obtained by enzyme digestion and enrichment of DNA using the full-length gold ATAC library construction kit, as detected by a 4150 TapeStation. Detailed Implementation

[0016] This invention provides a method for constructing an ATAC-seq library using quinoa microspores, comprising the following steps: 1) Select unopened quinoa flower buds, extract quinoa microspores, wash with buffer solution to obtain washed microspores; 2) Mix the washed microspores with Tn5 enzyme digestion buffer to obtain a mixture, then terminate the reaction to obtain the enzyme digestion solution; 3) Recover nucleic acids from the enzyme digestion solution using magnetic beads to obtain the ATAC-seq library; In this invention, unopened quinoa flower buds are selected, quinoa microspores are extracted, and washed with buffer solution to obtain washed microspores. The quinoa flower buds are obtained by collecting unopened flower buds when quinoa begins to flower and treating them at low temperature. The temperature of the low-temperature treatment is preferably 0~6℃, more preferably 2~5℃, and even more preferably 4℃. The treatment time is preferably 2~5 days, and more preferably 3~4 days. The unopened quinoa flower buds are preferably in the uninucleate stage of microspore development. The obtained quinoa flower buds are sterilized by soaking them in a sodium hypochlorite solution. The mass concentration of the sodium hypochlorite solution is preferably 5%~15%, more preferably 8%~12%, and even more preferably 10%. The soaking time is 5~15 min, more preferably 8~12 min, and even more preferably 10 min. The soaking temperature is preferably 20~30℃, more preferably 23~26℃, and even more preferably 25℃. The ratio of quinoa flower buds to sodium hypochlorite solution is preferably 80~12:10~30 mL, more preferably 90~110:15~25 mL, and even more preferably 100:20 mL.

[0017] In this invention, the method for extracting quinoa microspores is as follows: the extract is mixed with quinoa flower buds, chopped, filtered, and centrifuged to obtain quinoa microspores. The preferred ratio of quinoa flower buds to extract is 80-120 buds: 10-14 ml, more preferably 90-110 buds: 11-13 ml, and even more preferably 100 buds: 12 ml. The chopping is preferably performed using a high-speed disperser with a rotation speed of 1000-3000 rpm, more preferably 1500-2500 rpm, and even more preferably 23000 rpm. The rotation time is preferably 5-15 s, more preferably 8-12 s, and even more preferably 10 s. The filtration is preferably performed using a 200-500 mesh sieve, more preferably 250-400 mesh, and even more preferably 300 mesh. The centrifugation speed is preferably 300-1000 rpm, more preferably 400-800 rpm, and even more preferably 500 rpm. The centrifugation time is preferably 3-8 seconds. The extract is preferably taken at 4-6 min, and even more preferably at 5 min; the extract preferably contains the following concentrations of components: The concentration of mannitol is preferably 40-80 g / L, more preferably 50-70 g / L, and even more preferably 60 g / L; The concentration of calcium chloride is preferably 0.5~2 g / L, more preferably 0.7~1.5 g / L, and even more preferably 1.1 g / L; The concentration of MES is preferably 0.5~2 g / L, more preferably 0.7~1.5 g / L, and even more preferably 0.976 g / L; The pH of the extract is preferably 5.0 to 6.5, more preferably 5.5 to 6.0, and even more preferably 5.8; In this invention, quinoa microspores are extracted and washed with a buffer solution to obtain washed microspores. The buffer solution is M1 buffer [Zhao et al., Genome-wide MNase hypersensitivity assay unveils distinct classes of open chromatin associated with H3K27me3 and DNA methylation in Arabidopsis thaliana, Genome Biol 21, 24 (2020).]. The volume ratio of the buffer solution to quinoa microspores is preferably 1:1 to 5, more preferably 1:2 to 4, and even more preferably 1:3. The centrifugation speed during washing is preferably 500 to 1500 rpm, more preferably 800 to 1200 rpm, and even more preferably 1000 rpm. The washing time is preferably 1 to 3 min, more preferably 1.5 to 2.5 min, and even more preferably 2 min. The number of washes is preferably 1 to 5, more preferably 2 to 4, and even more preferably 3.

[0018] In this invention, the washed microspores are mixed with a Tn5 enzyme digestion buffer to obtain a mixture. The mixing temperature is preferably 35-40°C, more preferably 36-38°C, and even more preferably 37°C. The ratio of washed microspores to the Tn5 enzyme digestion buffer is preferably 3000-7000:20-50 μl, more preferably 4000-6000:25-40 μl, and even more preferably 5000:30 μl. The Tn5 enzyme digestion buffer contains the following components at the following final concentrations: The sucrose concentration is preferably 0.01~0.2 mg / mL, more preferably 0.05~0.15 mg / mL, and even more preferably 0.1 mg / mL; The Tris-HCl is preferably 0.03~0.07 M, more preferably 0.04~0.06 M, and even more preferably 0.05 M; The MgCl2 is preferably 2~6 mM, more preferably 3~5 mM, and even more preferably 4 mM; The preferred concentration of CaCl2 is 0.5~2 mM, more preferably 0.7~1.5 mM, and even more preferably 1 mM; The Tn5 enzyme is preferably 5~15 µM, more preferably 8~12 µM, and even more preferably 10 µM.

[0019] In this invention, after obtaining the mixture, the reaction is terminated to obtain the enzyme digestion solution; the termination is to mix the digestion buffer with the mixture; the mixing temperature is preferably 45~55℃, more preferably 48~52℃, and even more preferably 50℃; the mixing time is preferably 20~40 min, more preferably 25~35 min, and even more preferably 30 min.

[0020] In this invention, magnetic bead recovery involves mixing the enzyme digestion solution with magnetic beads, with the mixing time preferably being 3 to 10 minutes, more preferably 4 to 7 minutes, and even more preferably 5 minutes.

[0021] In this invention, after recycling with magnetic beads, the process further includes washing with alcohol. The concentration of alcohol is preferably 60% to 100%, more preferably 70% to 90%, and even more preferably 80%. The number of washing cycles is preferably 1 to 3, more preferably 2. The washing time for each cycle is preferably 20 to 40 seconds, more preferably 25 to 35 seconds, and even more preferably 30 seconds.

[0022] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0023] Example 1

[0024] Cut off the newly flowering quinoa inflorescences and treat them at 4°C for 2-5 days. Select flower buds at the uninucleate stage of microspore development, place them in Erlenmeyer flasks, sterilize with 10% sodium hypochlorite solution for 10 minutes, rinse 4 times with sterile water, and sterilize with 20 mL of sodium hypochlorite solution for every 100 flower buds. Place the sterile flower buds in 50 mL centrifuge tubes, about 100 flower buds per tube, and add 12 mL of extraction buffer (containing 60 g / L mannitol, 1.1 g / L calcium chloride, 0.976 g / L MES, pH 5.8, filtered and sterilized). Then, use a high-speed disperser to ultra-high speed on a clean bench to ultra-high speed. Filter the ultra-high speed suspension through a 300-mesh sieve, centrifuge the filtrate at 500 rpm for 5 minutes, and collect the microspores.

[0025] Microspores were placed in a 2ml centrifuge tube, 1ml of TE buffer and two steel beads were added, the mixture was shaken and ground, and then treated at 40Hz for 10s. Microspore cell wall fragments were observed under a microscope. Results are as follows: Figure 1 As shown, microspore cell walls have small pores, allowing the Tn5 enzyme to directly enter the cell and cleave DNA.

[0026] Example 2

[0027] When quinoa begins to flower, select unopened flower buds and extract microspores; Microspore ATAC-seq library construction was performed using a full-gold ATAC library construction kit (KP171); Approximately 5000 quinoa microspores were collected and added to 50 μl of 1×M1 buffer [0.1 M PBS (pH 7.0), 10 ml; 5 M NaCl, 2 ml; Beta-mercaptoethanol, 80 μl; Hexylene glycol, 11.35 ml; ddH2O to a final volume of 100 ml]. The mixture was centrifuged at 500 rpm for 5 min and washed twice to obtain the washed microspores.

[0028] Add the washed microspores to 30 μl of the fragmentation mix [26 μl buffer (50% sucrose, 2 ml 1 M Tris-HCl, pH 7.5, 500 μl 1 M MgCl2, 40 μl 1 M CaCl2, 10 μl); Tn5-50 Enzyme Mix, 4 μl]; incubate at 37°C for 30 minutes; add 10 μl of 4×Digestion Buffer to terminate the fragmentation reaction, and incubate at 50°C for 30 minutes.

[0029] Add 80 μl of 2× magnetic beads to the enzyme digestion reaction system and incubate at room temperature (16–26 °C) for 5 minutes. Place the centrifuge tube on a magnetic rack and incubate at room temperature until the solution becomes clear (approximately 5 minutes), then discard the supernatant. Keeping the centrifuge tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30 seconds, then discard the supernatant. Repeat once. Keeping the centrifuge tube on the magnetic rack, allow the magnetic beads to air dry at room temperature. Remove the centrifuge tube from the magnetic rack and add 21 μl of Library Elution Buffer. Mix well and incubate at room temperature for 3 minutes. Place the centrifuge tube on a magnetic rack and incubate at room temperature for 2 minutes, then carefully aspirate 20 μl of elution buffer into a clean PCR tube.

[0030] Take 2 μl of elution buffer from a PCR tube containing elution buffer and use a 4150 TapeStation to detect DNA fragment size. Results are as follows: Figure 2As shown, the DNA fragments are concentrated in the range of 100-300 bp, which is consistent with the distance between nucleosomes in open chromatin regions (approximately 200 bp), indicating that the DNA fragments digested by Tn5 enzyme were successfully enriched.

[0031] Comparative Example 1

[0032] DNA enriched using the full-gold ATAC library construction kit was added to microspores with 50 μl of 1×PBS, centrifuged at 500 rpm for 5 min, and washed twice. Microspores were then added to 30 μl of a fragmentation mix (4×Insertion Buffer, 7.5 μl; Insertion Enhancer 1, 0.3 μl; Insertion Enhancer 2, 0.3 μl; Tn5-50 Enzyme Mix, 4 μl; Nuclease-free Water, 17.9 μl), and incubated at 37°C for 30 min. The fragmentation reaction was terminated by adding 10 μl of 4×Tn5 Digestion Buffer, and incubated at 50°C for 30 min.

[0033] Add 80 μl of 2× magnetic beads to the enzyme digestion reaction system and incubate at room temperature for 5 minutes. Place the centrifuge tube on a magnetic rack and incubate at room temperature until the solution becomes clear (approximately 5 minutes), then discard the supernatant. Keeping the centrifuge tube on the magnetic rack, add 200 μl of freshly prepared 80% ethanol to the tube, incubate at room temperature for 30 seconds, then discard the supernatant. Repeat once. Keeping the centrifuge tube on the magnetic rack, allow the magnetic beads to air dry at room temperature. Remove the centrifuge tube from the magnetic rack and add 21 μl of Library Elution Buffer. Mix well and incubate at room temperature for 3 minutes. Place the centrifuge tube on a magnetic rack and incubate at room temperature for 2 minutes, then carefully aspirate 20 μl of elution buffer into a clean PCR tube.

[0034] Take 2 μl of elution buffer and analyze the DNA fragment size using a 4150 TapeStation. Results are as follows: Figure 3 As shown, no DNA fragments were found, indicating that the DNA fragments could not be successfully enriched using the full-size gold ATAC library construction kit.

[0035] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. A method for constructing an ATAC-seq library using quinoa microspores, characterized in that, Includes the following steps: 1) Select unopened quinoa flower buds, extract quinoa microspores, wash with buffer solution to obtain washed microspores; 2) Mix the washed microspores with Tn5 enzyme digestion buffer to obtain a mixture, then terminate the reaction to obtain the enzyme digestion solution; 3) Recover nucleic acids from the enzyme digestion solution using magnetic beads to obtain the ATAC-seq library; The Tn5 enzyme digestion buffer contains the following components at the following concentrations: Sucrose 0.1~0.3 mg / mL, Tris-HCl 0.01~0.1 M, MgCl2 2~6 mM, CaCl2 0.5~2 mM.

2. The method according to claim 1, characterized in that, Step 1) The method for extracting quinoa microspores includes the following steps: S1. When quinoa begins to flower, take the unflowered quinoa flower buds, treat them at 0~6℃ for 2~6 days, and then sterilize them to obtain sterile flower buds; S2. After extracting the microspores from the flower buds with the extract, centrifuge and discard the supernatant to obtain quinoa microspores; The extract contains the following components at the following concentrations: 40-80 g / L mannitol, 0.5-2 g / L calcium chloride, 0.5-2 g / L LMES, and pH 5.0-6.

5.

3. The method according to claim 2, characterized in that, The sterilization process in step S1 includes soaking the flower buds in a 5% to 20% sodium hypochlorite solution and then washing them 2 to 6 times. The ratio of quinoa flower buds to sodium hypochlorite solution is 80-12:10-30 mL; The soaking time is 5-20 minutes, and the soaking temperature is 20-30℃.

4. The method according to claim 1, characterized in that, Step 1) After washing, centrifuge and discard the supernatant; The cleaning is performed 1 to 5 times; The centrifugation speed is 5000~15000 rpm, and the centrifugation time is 1~3 min.

5. The method according to claim 1, characterized in that, Step 2) The ratio of the cleaned microspores to the Tn5 enzyme digestion buffer is 3000~7000:20~50 μL.

6. The method according to claim 5, characterized in that, Step 2) The mixing time is 20~40 min, and the mixing temperature is 35~40℃.

7. The method according to claim 1, characterized in that, Step 2) The termination step involves mixing the digestion buffer solution with the mixture. The mixing temperature is 45~55℃; The mixing time is 20-40 minutes.

8. The method according to claim 1, characterized in that, Step 3) The recovery using magnetic beads involves mixing the enzyme digestion solution with magnetic beads for 3-10 minutes.

9. The method according to claim 8, characterized in that, Step 3) The recovery process using magnetic beads also includes washing the magnetic beads with 60%~100% alcohol.

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