CN111733214A - High-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformation of frozen biological sample - Google Patents

High-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformation of frozen biological sample Download PDF

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CN111733214A
CN111733214A CN202010522547.3A CN202010522547A CN111733214A CN 111733214 A CN111733214 A CN 111733214A CN 202010522547 A CN202010522547 A CN 202010522547A CN 111733214 A CN111733214 A CN 111733214A
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李付广
郑武
葛晓阳
王晔
杨召恩
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Institute of Cotton Research of Chinese Academy of Agricultural Sciences
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Abstract

The invention discloses a high-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformation of a frozen biological sample. The invention provides a sample pretreatment method suitable for Hi-C high-throughput sequencing library construction, which comprises the following steps: freezing the biological sample to be tested, then heating up in a gradient way, and then fixing by crosslinking. The FS-Hi-C technology is not only suitable for fresh samples, but also suitable for frozen samples. The results of the invention show that the chromatin three-dimensional structures of the frozen replacement sample and the fresh sample are highly similar, and the quality of the Hi-C library is improved. Compared with the traditional Hi-C pretreatment method, the FS-Hi-C method has the advantages that the chromatin interaction, A/B component, TAD and the like are highly consistent, the quality of the constructed library by the FS-Hi-C method is better, the library capacity is increased, effective interaction data are more, and repeated data generated by PCR amplification are reduced. This technique reduces the requirement for sample timeliness. The invention provides an improved method for application of Hi-C in organisms.

Description

High-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformation of frozen biological sample
Technical Field
The invention relates to the technical field of chromosome conformation capture, in particular to a high-throughput sequencing and database building technology capable of efficiently capturing three-dimensional chromosome conformations of a frozen biological sample.
Background
The chromosome is a special structure existing in a cell nucleus, and according to the principle of 'structure determining function', the three-dimensional (3D) structure of the chromosome naturally becomes the basis for understanding the biological function of the chromosome, and the 3D structure of the genome plays an important role in the replication, damage repair and transcriptional regulation of DNA. By exploring the remote control function among chromatins, unknown target genes of the regulatory elements can be identified, and genes regulated by cis-regulatory elements, phenotypes regulated by eQTL, TAD recombination and gene expression regulated by A/Bcomparatent conversion are researched, however, the progress of chromosome 3D structure analysis is slow all the time due to the technical reasons.
In 2002, Dekker et al established 3C chromosome conformation capture (3C) technology, which is a chromosome conformation technology for studying chromosome and protein interaction and can analyze the correlation between gene loci with long linear distance. The 3C technology solves the spatial relationship of 2 DNA fragments with long straight line distance, namely the one-to-one relationship. The technology has milestone significance for the research of the field of chromosome DNA space structure, opens up a new era of the research of chromatin three-dimensional high-grade structure, and inspires a series of derivative technologies. The 4C and 5C technologies appeared in 2006, which solved the relationship of "one-to-many" and "many-to-many" of DNA fragments, respectively. Although these derivation techniques have advanced the understanding of the interrelationships between chromosome fragments, there is still no overall understanding of the chromosome 3D structure.
In recent years, with the development of increasingly sophisticated high throughput sequencing technologies, the acquisition of large-scale genomic information has become easier. In 2009, Dekker et al combined chromosome conformation capture with an increasingly mature high throughput sequencing technology and established the highest throughput Hi-C (highest-throughput chromosome conformation capture) technology for the first time. Hi-C is a high-throughput sequencing technology for analyzing the spatial conformation of chromosomes, can capture the spatial interaction between different gene loci in the whole genome range, and is helpful for researchers to understand the three-dimensional spatial structure of chromosomes, the interaction between chromosomes and the spatial regulation mechanism of gene expression. The Hi-C technology is modified on the basis of the 3C technology, biotin-labeled nucleotides are added, small fragments of DNA generated by subsequent shearing can be enriched, sequencing joints are added to two ends of the fragments, and then the results are compared and analyzed by adopting the latest sequencing means. The technology has the advantages of multiple steps, long consumed time, complex related reagent consumables and more space for improving and optimizing the whole process. These drawbacks of existing Hi-C high throughput sequencing limit the application of this technology to the promotion of functional genomic research, and there is a need for improvements to this technology that provide a new Hi-C high throughput sequencing library-building method that is efficient, convenient, economical and widely applicable. Chromosomal interactions play an important role in genome structure and gene regulation, and Hi-C is a powerful tool for studying the three-dimensional genome structure of species. However, the acquisition of native chromatin conformation requires fresh samples, which hinders the progress of three-dimensional genomic studies.
The solid state of water has three forms, including two crystal forms (hexagonal and cubic) and a glassy state. The glassy state is due to rapid freezing of the sample without time for crystals to form. The formation of crystals causes water to expand when frozen, but the glassy water does not expand after solidification, which makes the glassy water the only ideal frozen form for biological specimens. Based on the above principle, liquid nitrogen (LN2) is widely used for freezing and long-term preservation of biological samples. However, frozen specimens are rarely used for three-dimensional genomic studies. It is generally believed that the three-dimensional genome structure is affected by the sample after being removed from the liquid nitrogen solution during the temperature rise process. The freeze replacement (FS) technique is generally used to maintain the structure of single-and multi-cell animals and plants, and thus it is widely used to prepare samples for conventional optical, transmission and scanning electron microscopes.
Disclosure of Invention
The invention optimizes and improves the key steps of the conventional Hi-C technology which is complicated in sample preparation and not easy to control quality so as to facilitate standardization and quality control, provides more stable and reliable experimental results and promotes further wide application of the Hi-C technology.
In a first aspect, the invention claims a sample pretreatment method suitable for Hi-C high-throughput sequencing and library building.
The sample pretreatment method applicable to Hi-C high-throughput sequencing and library building, which is claimed by the invention, can comprise the following steps: the biological sample to be tested is frozen and then cross-linked and fixed.
Wherein the freezing step can be a low temperature treatment at-196 ℃.
In the invention, the freezing is to put the tested biological sample into liquid nitrogen for quick freezing and grind the sample into powder in the liquid nitrogen.
Wherein, the cross-linking fixation can be cross-linking fixation by using formaldehyde solution. Wherein, the formaldehyde solution can be 37 percent by volume of formaldehyde solution. When the crosslinking and fixing are carried out, the final concentration of formaldehyde in the system is 1 percent by volume.
Further, the process of pre-freezing and crosslinking the biological sample to be tested and gradient temperature rise is also included between the freezing and the crosslinking and fixing. The method can prevent the three-dimensional structural change generated in the temperature rising process after the sample is removed from the liquid nitrogen solution. The biological problem of how to use a low-temperature frozen storage sample for researching the three-dimensional structure of the chromosome is solved, the chromatin conformation in a living cell can be effectively maintained, and the Hi-C data quality of organisms is improved.
Wherein, the pre-freezing crosslinking is carried out in a pre-freezing crosslinking liquid, the pre-freezing crosslinking liquid is an ethanol solution containing 2 percent of water and 0.01 percent of formaldehyde, and the percent represents the volume percentage content.
Wherein the gradient temperature rise is 6h at-90 ℃, 6h at-60 ℃, 6h at-30 ℃ and 6h at 0 ℃. In the gradient temperature rise process, the temperature rises from-90 ℃ to-60 ℃ by 5 ℃ per hour, the temperature rises from-60 ℃ to-30 ℃ by 5 ℃ per hour, and the temperature rises from-30 ℃ to 0 ℃ by 5 ℃ per hour.
Still further, the method may comprise the step of subjecting the test biological sample to the following processes in sequence:
(A1) quick-freezing and grinding by using liquid nitrogen;
(A2) pre-freezing and crosslinking;
(A3) gradient heating;
(A4) centrifuging, adding NIbuffer and filtering;
(A5) formaldehyde crosslinking and fixing;
(A6) glycine terminates crosslinking;
(A7) the cell nuclei were recovered by centrifugation.
In step (a1), the test biological sample was snap frozen in liquid nitrogen and ground to a powder in liquid nitrogen.
In step (A2), the milled powder of (A1) was transferred to a pre-frozen cross-linked solution at-90 ℃ in ethanol containing 2% water, 0.01% formaldehyde,% expressed as volume%.
In step (A3), the gradient temperature rise can be specifically-90 ℃ for 6h, -60 ℃ for 6h, -30 ℃ for 6h, so as to replace the water in the cells with ethanol solution.
In the gradient heating process, the temperature is increased from-90 ℃ to-60 ℃ by 5 ℃ per hour in a gradient manner, and the temperature is increased from-60 ℃ to-30 ℃ by 5 ℃ per hour in a gradient manner.
In step (a4), the recipe for NIbuffer is as follows: 20mM Hepes pH8, 250mM sucrose,1mM magnesium chloride, 5mM potassium chloride, 40% glycerol by volume, 0.25% Triton X-100 by volume, 0.1mM PMSF, 0.1% beta-mercaptoethanol by volume, 1/5 volume fraction of cocktail. Wherein said cocktail is a protease inhibitor. In a specific embodiment of the present invention, the cocktail is a product of MCE, Inc. under the trade designation HY-K0010.
In step (A4), centrifuging (e.g. at 4 deg.C for 30s) to remove supernatant (alcohol solution), adding pre-cooled (ice-cooled) NIbuffer, washing three times as required, and washing 1-2g (e.g. 2g) of the biological sample (or 2 × 10 g)6Individual drosophila cell samples) 20mL of the NIbuffer pre-chilled (pre-chilled on ice) was added to form a suspension, then gently shaken for 15min, then Miracloth (Millipore, cat #: 475855) filtering (optionally twice), collecting the filtrate, and centrifuging (e.g. 3000g at 4 deg.C for 15 min).
Further, in the step (a5), an aqueous formaldehyde solution having a concentration of 37% by volume was added to the supernatant obtained by the centrifugation in (a 4). Wherein the final concentration of formaldehyde in the system is 1% volume percentage content. And when the crosslinking and fixing are carried out, the crosslinking temperature is room temperature, and the time is 8 min.
Further, in step (A6), the crosslinking can be terminated by adding a glycine solution (solvent is water) at a concentration of 2.5M.
Further, in step (A7), the centrifugation may be 1500g at 4 ℃ for 5 min. In a second aspect, the invention claims a Hi-C high throughput sequencing and library building method.
The Hi-C high-throughput sequencing and database building method claimed by the invention can comprise the following steps: pre-treating a test biological sample by the method described above; the treated samples were then subjected to Hi-C high throughput sequencing for pooling.
In a third aspect, the invention claims a Hi-C high throughput sequencing method.
The Hi-C high-throughput sequencing method claimed by the invention can comprise the following steps: pre-treating a test biological sample by the method described above; then, performing Hi-C high-throughput sequencing on the processed sample to build a library; and finally performing Hi-C high-throughput sequencing.
In the above three aspects, the biological sample may be a cell or tissue, such as fresh cells or tissue.
In the above three aspects, the organism is a plant (such as cotton, soybean or radish) or an animal (such as fruit fly).
In one embodiment of the invention, the biological sample is specifically callus or Drosophila cell line of cotton.
In a fourth aspect, the invention claims the use of any one of:
(B1) use of a method as hereinbefore described in the first aspect in Hi-C high throughput sequencing pooling;
(B2) use of the method described in the first and second aspects hereinbefore in Hi-C high throughput sequencing.
The invention creatively develops a simple freezing replacement Hi-C technology (Frozen subitute-Hi-C), and the method is not only suitable for fresh samples, but also suitable for Frozen samples stored in liquid nitrogen. The technology applies a freezing replacement (Frozen Substitet) technology to Hi-C sample preparation of a liquid nitrogen (LN2) Frozen sample, and avoids three-dimensional structural change during temperature rising after the sample is removed from a liquid nitrogen solution. The method effectively expands the application range of the Hi-C sample from a fresh sample to a frozen sample, and solves the problems that the sample is difficult to sample and cannot be transported in a long distance. It is noteworthy that the present invention grinds the sample in liquid nitrogen before the freeze replacement (FS) step in plants, which can remove plant cell walls that adversely affect plant cross-linking. The use of cryo-replacement Hi-C replaces the liquid water inside the sample cells with ethanol, so that the frozen sample maintains an unchanged chromatin conformation during warming. The new method is examined in drosophila, cotton, soybean and radish, and the observation of a transmission electron microscope shows that the chromatin structures of a frozen replacement (FS) sample and a fresh sample are highly similar. The chromatin interaction, A/B component and TAD aspects showed a high degree of consistency compared to traditional Hi-C. But the library constructed by the FS-Hi-C method has better quality, increased storage capacity, more effective interaction data and reduced repeated data generated by PCR amplification. The technology reduces the requirement on the timeliness of the sample, and facilitates the collection and storage of the sample. The invention breaks through the limitation that a Hi-C test needs a fresh sample, improves the quality of library data, maintains chromatin conformation, and paves a way for further exploring gene regulation and three-dimensional genome structure.
The technical key point of the invention is that a sample is ground by using liquid nitrogen quick freezing, then pre-crosslinking and gradient heating are carried out, and then crosslinking and fixing are carried out. The invention evaluates the library quality of freezing replacement Hi-C (FS-Hi-C), and finds that in cotton and fruit flies, the three-dimensional conformation of a fresh sample is similar to that of a sample subjected to gradient temperature rise after freezing; and the pretreatment of the sample is carried out by using a freezing replacement mode, so that the data quality of the Hi-C library of cotton and drosophila can be improved. The invention breaks the limitation that a fresh sample must be used in the Hi-C test, and provides wider prospect for the application of Hi-C in biological research.
The invention provides a Hi-C experimental process suitable for organisms such as fruit flies, cotton, soybeans and radishes, solves the technical problems of low cross-linking quality of biological cells and poor establishment of a biological sample library through an innovative and optimized pretreatment method, and obviously improves the effective data volume of the established Hi-C library.
Compared with the prior art, the invention has the following advantages:
(1) in biological experiments, frozen replacement of Hi-C (FS-Hi-C) did not affect the three-dimensional conformation of the chromosome.
(2) In biological experiments, freezing to replace Hi-C (FS-Hi-C) significantly improved the quality of Hi-C library.
(3) In a biological experiment, freezing replacement of Hi-C (FS-Hi-C) effectively expands the application range of a Hi-C sample from a fresh sample to a frozen sample, and solves the problems that the sample is difficult to sample and cannot be transported for a long distance.
(4) In cotton and Drosophila samples, frozen replacement of Hi-C (FS-Hi-C) had no significant effect on chromosome 3D structure.
The FS-Hi-C technology provided by the invention obviously improves the effective data volume of the constructed Hi-C library. Is suitable for the construction of Hi-C libraries of organisms.
Drawings
FIG. 1 is a block diagram of the pretreatment process of the FS-Hi-C high-throughput sequencing and database building method for organisms according to the present invention.
FIG. 2 is a schematic flow chart of the FS-Hi-C method applicable to organisms according to the present invention.
FIG. 3 shows agarose gel electrophoresis measurements of the genomes obtained from two different pre-treatments of the plant samples in the first batch of experiments (small scale sequencing) of example 1. 1 and 8: DNA marker; 2: genome 3 extracted by traditional methods: the genome extracted by the method of the invention; 4: the result of the enzyme digestion (endonuclease DpnII) by the traditional method; 5: the enzyme digestion (endonuclease DpnII) result of the method is obtained; 6: connecting the results after enzyme digestion by the traditional method; 7: the method of the invention links the results after enzyme digestion.
FIG. 4 is a graph of Hi-C matrices constructed at 1Mb resolution using HiCPro software in the first batch of experiments (small scale sequencing) of example 1. In the figure, H and MH are conventional processes and F and CF are the processes of the present invention.
FIG. 5 is a heat map of the cotton chromosome interaction between the cryo-replacement Hi-C (FS-Hi-C) and the conventional Hi-C method treatment in the second batch of experiments (deep sequencing) of example 1.
FIG. 6 is a graph comparing the distribution patterns of frozen replacement Hi-C (FS-Hi-C) and A/B components in cotton treated by the conventional Hi-C method in the second lot of experimental deep sequencing of example 1.
FIG. 7 is a comparison of the topologically related domains (TADs) of cryo-substituted Hi-C (FS-Hi-C) and cotton treated by the traditional Hi-C method in the second trial depth sequencing of example 1.
FIG. 8 is a graph of the change in chromatin structure in nuclei under different treatments in example 1: a. b-f processing modes in the graph; b. chromatin structure of fresh cotton leaves after high pressure freezing and cryo-replacement treatment (HPF-FS-TEM); c. replacing the chromatin structure of the processed karyon by high-pressure freezing and freezing after freezing, grinding and directly heating; d. performing high-pressure freezing and freezing replacement on the chromatin structure of the cell nucleus extracted by the traditional Hi-C method; e. chromatin structure of FS-Hi-C treated cell nucleus extracted without gradient heating process after high pressure freezing and freezing replacement; f. freezing replaces chromatin structure of Hi-C (FS-Hi-C) extracted nuclei after high pressure freezing and freezing replacement.
FIG. 9 is a heat map of the chromosomal interactions of drosophila treated with the frozen replacement Hi-C (FS-Hi-C) and the traditional Hi-C method in example 2.
FIG. 10 is a comparison of distribution patterns of A/Bcomparatents in Drosophila of the frozen replacement Hi-C (FS-Hi-C) and the conventional Hi-C method in example 2.
FIG. 11 is a comparison of topologically related domains (TADs) in Drosophila of example 2 with the frozen replacement of Hi-C (FS-Hi-C) and the traditional Hi-C method.
Detailed Description
The pretreatment flow chart of the FS-Hi-C high-throughput sequencing and database building method suitable for plants is shown in figure 1, and the following treatments are sequentially carried out on a biological sample to be tested: liquid nitrogen quick freezing and grinding, pre-freezing and crosslinking, gradient heating, centrifuging and filtering by NIBbuffer, crosslinking and fixing by 37 percent formaldehyde, and centrifuging and recovering cell nuclei. A schematic flow chart of the FS-Hi-C method applicable to organisms in the invention is shown in FIG. 2.
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention.
The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.
Pre-freezing the crosslinking liquid: alcohol solution containing 2% of water and 0.01% of formaldehyde,% represents volume percentage.
NIbuffer: 20mM Hepes pH 8; 250mM sucrose; 1mM MgCl25mM KCl, 40% (v/v) glycerol, 0.25% (v/v) Triton X-100,0.1mM PMSF, 0.1% (v/v) β -mercaptoethanol, 1/5 volume fraction of cocktail (protease inhibitor, product of MCE, cat. No. HY-K0010).
1.2 × NEBuffer 2: 10 XNEBuffer 2 is a product of NEB company, the product number: B7002S. Diluting as required.
NEB buffer 3.1: NEB buffer3.1 is a product of NEB company, and the product number is: B7203.
10 × NEBuffer 2: NEB company, cat no: B7002S.
1 × NEBuffer 2: 10 XNEBuffer 2 is a product of NEB company, the product number: B7002S. Diluting as required.
Example 1 Hi-C high throughput sequencing of Cotton
In this example, two batches of experiments were performed with callus from cotton tissue culture as the subject, wherein the first batch was subjected to small-scale sequencing (10g data volume, two small data evaluations) of the Hi-C libraries of the two methods to evaluate the quality of the libraries, and the Hi-C library from the new Hi-C method was found to be much higher in the effective data ratio than the conventional method; in the second batch, we performed deep sequencing of the libraries (three each) from both methods of cotton with genomic coverage of approximately 75X, 50X and 30X (FS-Hi-C); 50X, 50X and 30X (conventional Hi-C). As a result, FS-Hi-C remarkably improves the effective data proportion of the Hi-C library and greatly reduces the repetitive sequence proportion caused by PCR amplification in the library.
First, first batch test
(I) sample pretreatment
The method of the invention：
1. Fresh cotton callus was blotted dry with filter paper and 4g was snap frozen in liquid nitrogen and ground to a powder.
2. Transferring the ground cotton callus powder to pre-freezing cross-linking liquid at-90 deg.C, slowly raising the temperature in gradient from-90 deg.C to-60 deg.C to-30 deg.C to 0 deg.C, each temperature gradient lasting 6h (raising the temperature from-90 deg.C to-60 deg.C is raising the temperature 5 deg.C per hour, raising the temperature from-60 deg.C to-30 deg.C is raising the temperature 5 deg.C per hour, raising the temperature from-30 deg.C to 0 deg.C is raising the temperature 5 deg.C per hour). The water in the cells is replaced by an ethanol solution.
3. Centrifuging at 4 deg.C for 30s at 3000g, removing ethanol solution, washing with ice-precooled NIbuffer solution three times, adding precooled (ice-precooled) NIbuffer in an amount of 20mL per 2g cotton callus to form suspension, and gently shaking for 15 min.
4. The suspension was filtered twice with Miracloth (Millipore, cat # 475855), the filtrate was collected, centrifuged at 4 ℃ for 15min at 3000g, 37% by volume of aqueous formaldehyde was added to the supernatant obtained by centrifugation in an amount of 800. mu.L per 2g of cotton callus (so that the final concentration of formaldehyde in the system was 1% by volume), fixed by crosslinking at room temperature for 8min, and terminated by addition of glycine (solvent is water) at a concentration of 2.5M.
5. Cell nuclei were recovered by centrifugation at 1500g and 4 ℃ for 5 min.
Conventional methods：
1. Collecting 4g to 10g of fresh tissue sample;
2. add 45mL PBS (or sugarless MS) and 1.25mL 37% formaldehyde solution (sigma) in a fume hood (final concentration 1%) to each 50mL centrifuge tube;
3. each 2-3g sample was cut into 2 mm. times.3 mm. times.10 mm pieces with a sharp blade, placed in the above 50mL centrifuge tube, evacuated at room temperature to-60 to-80 Kpa, kept for 10min, and then returned to atmospheric pressure. Repeating the above steps for three times (standard: sample sinking);
4. the crosslinking reaction was terminated by adding 2.5mL of 2.5M glycine (stored at 4 ℃ C.) (final concentration: 0.125M), followed by shaking for 5min to terminate the reaction.
5. Filtrate off the liquid, ddH2Cleaning for 3 times, drying the liquid on the surface of the sample by using absorbent paper, quickly freezing by using liquid nitrogen, storing at-80 ℃, and transporting by using dry ice.
6. After crosslinking, the sample was ground well into a powder with liquid nitrogen and transferred to a 50mL centrifuge tube containing 10mL ice-chilled NIbuffer (NIbuffer, 20mM Hepes pH8, 250mM Cross, 1mM MgCl)25mM KCl, 40% (v/v) glycerol, 0.25% (v/v) Triton X-100,0.1mM PMSF, 0.1% (v/v) β -ME,1/5cocktail), gently shaken on ice for 15 min.
7. The suspension was filtered twice through miracloth (Millipore), the filtrate collected in a 50mL centrifuge tube and centrifuged at 3000g for 15min at 4 ℃.
(II) library building process
1. The supernatant was discarded and the pellet was resuspended in 2mL of NIbuffer and centrifuged at 1900g for 5min at 4 ℃.
2. Repeat step 1 once.
3. The supernatant was discarded. Resuspend in 1mL of 1.2 XNEBuffer 2, centrifuge at 1900g for 5min at 4 ℃.
4. The supernatant was discarded and resuspended in 500. mu.L of 1.2 XNEBuffer 2.
5. 7.5 μ L of 20% (w/v) SDS was added. Incubate at 65 ℃ for 40 min. Incubate at 37 ℃ for 20min with a shaker at 800 rpm.
6. 50 μ L of 20% (v/v) Triton X-100 was added and incubated at 37 ℃ for 1h with a shaker at 800 rpm.
7. Add 50. mu.L NEB buffer3.1, 10. mu.L DpnII and spin overnight at 37 ℃.
8. DpnII was inactivated at 62 ℃ for 20 minutes.
9. Biotin-labeled DNA (1.5. mu.L of 10mM dATP, 1.5. mu.L of 10mM dGTP, 1.5. mu.L of 10mM dTTP, 37.5. mu.L of 0.4mM biotin-14-dCTP) and 10. mu.L of 5U/. mu.L Klenow enzyme-filled DNA were added, carefully mixed and incubated at 37 ℃ for 45 min.
10. The reaction mixture was removed and placed on ice, and 86. mu.L of 10% (w/v) SDS was added to terminate the reaction. Incubate at 65 ℃ for 30min, then quickly place on ice.
11. The DNA was filled in, and 7.61mL of a loop reaction system (745. mu.L of 10% (v/v) Triton X-100,745. mu.L of 10 × ligation buffer (500mM Tris-HCl pH 7.5,100mM MgCl) was added to ice2100mM DTT), 80. mu.L of 10mg/mL BSA, 80. mu.L of 100mM ATP and 5.96mL ddH2O) to a 15mL centrifuge tube, transfer chromatin to the 15mL centrifuge tube.
12. 50U T4 DNA ligase was added thereto, the mixture was inverted and mixed, and reacted at 16 ℃ for 4 hours to form a loop of DNA.
13. Add 50. mu.L 10mg/mL proteinase K and digest overnight at 65 ℃. Then, 50. mu.L of 10mg/mL proteinase K was added and digested at 65 ℃ for 2 hours.
14. Cooled to room temperature and transferred to a 50mL centrifuge tube. DNA was purified by phenol extraction. Add 10mL phenol pH8.0 and vortex for 2 min. Centrifuge at 1500g for 10min and transfer the aqueous phase to a new 50mL centrifuge tube.
15. Re-extraction and purification by phenol chloroform (1: 1 volume), vortexing and shaking for 2min, centrifuging at 22000g for 5min, and transferring the aqueous phase to a new 50mL centrifuge tube.
16. Adding 2.5 times volume of pure alcohol to precipitate DNA, centrifuging at 22000g for 5min, and discarding the supernatant.
17. The DNA pellet was dissolved by adding 450. mu.L of TE (pH 8.0) and transferred to a 2mL centrifuge tube.
18. Add 500. mu.L phenol chloroform (1: 1 vol), shake for 1min, centrifuge at 22000g for 5min, transfer the aqueous phase to a new tube. After two purifications, 0.1 volume of NaOAc and 2 volumes of absolute alcohol were added, incubated at-80 ℃ for 30min and the supernatant was centrifuged off.
19. The DNA was washed by adding 1mL of 70% ethanol and centrifuged at 22000g for 5 min. The supernatant was discarded.
20. Add 25. mu.L of TE to resuspend the DNA. Add 1. mu.L of 1mg/mL RNase A and digest for 30min at 37 ℃.
21. Quality inspection: a5. mu.L sample was taken for detection on 1% agarose gel. And the sample with obvious main band is unqualified and needs to be treated and then is subjected to enzyme digestion. If a diffuse band is present, the sample continues to follow-up.
FIG. 3 shows the agarose gel detection results of the genomic samples extracted by the conventional method and the method of the present invention, the samples after the genomic digestion, and the samples after the digestion and ligation. The result shows that the method of the invention presents a dispersion strip after enzyme digestion, which indicates that the pretreatment of the sample is qualified, and the result is consistent with the treatment result of the traditional method, and the subsequent experiment can be carried out.
22. The unclycled biotin-labeled DNA was removed using the exonuclease activity of T4 DNA polymerase. Mu.g of library was added to 100. mu.L of a system containing 1. mu.L of 10mg/mL BSA, 10. mu.L of 10 XNEBuffer 2, 1. mu.L of 10mM dATP, 1. mu.L of 10mM dGTP and 5 Units T4 DNA polymerase, and incubated at 16 ℃ for 4 hours.
23. The reaction was stopped by adding 2. mu.L of 0.5M EDTA, pH8.0, and the DNA was purified by phenol chloroform (1: 1, volume ratio)/ethanol precipitation. 100 μ L ddH2O resuspend DNA.
24. The DNA was disrupted to around 200-700bp using an ultrasonic DNA disruptor (Covaris M220).
25. The ends were repaired by adding 14. mu.L of 10 × ligation buffer, 14. mu.L of 2.5mM dNTPmix, 5. mu. L T4 DNApolymerase, 5. mu. L T4 nucleotide kinase, 1. mu.L of Klenow DNA polymerase and 1. mu.L of water and incubating at 20 ℃ for 30 min.
26. DNA purification was performed by DNA purification kit (Qiagen). After elution twice with 15. mu.L of low-salt TE (TLE:10mM Tris pH8.0,0.1mM EDTA), the DNA was added with A and incubated for 20 minutes at 37 ℃ with 5. mu.L of 10 XNEBuffer 2, 10. mu.L of 1mM dATP, 2. mu.L of water and 3. mu.L of Klenow (exo-).
27. The reaction was stopped by incubation at 65 ℃ for 20min and quickly placed on ice. Concentrate to 20 μ L in vacuo.
28. The DNA was recovered by running on a 1.5% agarose gel 80-90v for 3.5 hours. The DNA of 300-500bp is selected, cut and recovered by a gel recovery kit. Elute with 50 μ L TLE. The eluates were pooled to 300. mu.L. The total amount of DNA was calculated by Qubit.
29. The following experiments are recommended to be performed in low DNA adsorption tubes (DNA Lobind tubes).
30. Biotin DNA enrichment was performed by the magnetic bead method, with detailed reference to kit instructions (AgencourtAmpure areas, Beckman). Specifically, the method comprises the following steps:
A. biotin pull-down beads were prepared by washing 150. mu.L of resuspended streptavidin magnetic beads twice with 400. mu.L of LTween buffer (TB: 5mM Tris-HCl pH8.0, 0.5mM EDTA, 1M NaCl, 0.05% Tween).
B. The magnetic beads were resuspended in 300. mu.L of 2X buffer without Tween (2 XTB: 10mM Tris-HCl pH8.0, 1mM EDTA, 2M NaCl) and 300. mu. LHi-C DNA obtained in step 28 was added. The mixture was incubated at room temperature for 15 minutes while rotating, and then biotin-labeled Hi-C DNA was bound to streptavidin-containing magnetic beads.
C. Streptavidin magnetic beads bound to DNA were recovered with a magnetic particle concentrator and the supernatant was discarded. The beads were washed sequentially with 400. mu.L of 1 XNTB (5mM Tris-HCl pH8.0, 0.5mM EDTA, 1M NaCl) and 100. mu.L of 1 Xligation buffer. The beads were then resuspended in 50. mu.L of 1 × ligation buffer, and the mixture was transferred to a new tube.
D. The magnetic beads were resuspended in Gibson kit buffer and digested for 10min at 37 ℃ with T5 exonuclease (ref: enzymatic assembly of DNA molecules up to sectional cloned kits).
E. The ends were repaired by adding 14. mu.l of 10 Xligation buffer, 14. mu.l of 2.5mM dNTPmix, 5. mu. l T4 DNApolymerase, 5. mu. l T4 nucleotide kinase, 1. mu.l Klenow DNA polymerase and 1. mu.l water and incubating at 20 ℃ for 30 min.
F. DNA purification was performed by DNA purification kit (Qiagen). After elution twice with 15. mu.l of low-salt TE (TLE:10mM Tris pH8.0,0.1mM EDTA), the DNA was added with A and incubated for 20 minutes at 37 ℃ with 5. mu.l of 10 XNEBuffer 2, 10. mu.l of 1mM dATP, 2. mu.l of water and 3. mu.l of Klenow (exo-).
The reaction was stopped by incubation at G.65 ℃ for 20min and quickly placed on ice.
H. The total amount of DNA calculated in step 28 was used as the input for biotin pulldown and divided by 20 to estimate the total amount of Hi-C DNA that was pulled down and available for linker ligation and sequencing. The adaptors were added at a rate of 6 picomoles of Illumina double-ended linker per microgram of Hi-C DNA available for ligation. 1200 units of T4 DNA ligase was used to ligate the linker to the DNA. Incubate at room temperature for 2 hours.
I. The magnetic beads bound to the Hi-C DNA were recovered and washed twice with 400. mu.L of 1 XTB, thereby removing the excess double-ended linker.
J. The beads were washed sequentially with 200. mu.L of 1 XNTB, 200. mu.L and 50. mu.L of 1 XNEBuffer. After the last wash, it was resuspended in 50. mu.L of 1 XNEBuffer 2 and transferred to a new tube.
K. To determine the appropriate number of cycles to generate enough PCR products to be sequenced, PCR experiments were performed at 6, 9, 12 and 15 cycles, respectively. The optimal number of cycles was determined by PCR reactions on a 5% polyacrylamide gel and staining with Sybr Green, ensuring that no bands were present and that bands appeared in the 400-base pair segment, since this is the approximate fragment length after ligation of the Hi-C DNA to the linker.
L. Large scale PCR was performed at the optimal number of PCR cycles to amplify the remaining Hi-C DNA library bound to magnetic beads. The PCR products were collected separately and the magnetic beads were recovered. 1% of the PCR product was placed on a gel as a control, and the remaining PCR product was purified using 1.8 volumes of Ampure beads.
M. eluting the purified PCR product by 50 mu L of 1 XTLE buffer solution, namely the constructed Hi-C library.
Through the above-described procedures, two Hi-C libraries (designated as T1 and T2) obtained by the conventional method and two Hi-C libraries (designated as F1 and F2) obtained by the method of the present invention were obtained.
(III) Hi-C high throughput sequencing
Pair-end sequencing (paired-end sequencing) was performed using an Illumina: HiSeq X Ten sequencer.
(IV) quality identification of Hi-C library
The high throughput sequencing data of 4 Hi-C libraries obtained by the conventional and inventive methods were evaluated. Quality control results generated by the accepted software HiCPro are shown in table 1 (small scale sequencing). The results show that, compared with the traditional method, the logarithmic ratio of the Reads with high quality obtained by the method is higher than that obtained by the traditional method; the proportion of Reads contaminated with the joint of the present invention is lower than in the conventional method; the logarithmic proportion of Reads of the genome is not compared at both ends of the method of the invention and is lower than that of the traditional method; the ratio of the Reads of the repetitive sequence generated in the PCR process to the logarithm of the Reads of different enzyme digestion fragments of both ends of the PE is far greater than that of the prior method. And (4) conclusion: the quality of the Hi-C library of the method is superior to that of the library built by the traditional method.
TABLE 1 quality control results of Hi-C library high throughput sequencing data of the present and conventional methods generated by the software HiCPro
Figure BDA0002532603870000091
Next, HiCPro software was used to construct a Hi-C matrix map at 1Mb resolution, as shown in FIG. 4. It can be seen that, at the genome-wide level, the rapid freezing cross-linking method of the invention has high similarity of three-dimensional chromatin conformations compared with the traditional method; refined to the level of each chromosome (a01), and the Hi-C heatmap correlation of the two showed that the DNA-DNA interaction intensity remained highly consistent in both treatment groups (flash freeze cross-linked and traditional methods treatment groups); in addition, chromatin component treated by both methods was also highly similar. This indicates that the rapid freeze-crosslinking treatment did not affect the three-dimensional chromatin conformation of cotton.
Second, second batch test
(I) sample pretreatment
The method of the invention：
1. Fresh cotton callus was blotted dry with filter paper and 4g was snap frozen in liquid nitrogen and ground to a powder.
2. Transferring the ground cotton callus powder to a pre-freezing cross-linking liquid at-90 ℃, and then slowly raising the temperature in a gradient way: 6h at 90 ℃ below zero; the temperature is between 90 ℃ below zero and 60 ℃ below zero for 6 hours (the temperature is raised by 5 ℃ per hour); 6h at 60 ℃ below zero; the temperature is between 60 ℃ below zero and 30 ℃ below zero for 6 hours (the temperature is raised by 5 ℃ per hour); 6h at-30 ℃; -30 ℃ to 0 ℃ for 6h (5 ℃ per hour); each temperature gradient lasted 6 h. The water in the cells is replaced by an ethanol solution.
3. Centrifuging at 4 deg.C for 30s at 3000g, removing ethanol, washing with ice-precooled NIbuffer solution three times, adding precooled (ice-precooled) NIbuffer in an amount of 20mL per 2g cotton callus to form suspension, and gently shaking for 15 min.
4. The suspension was filtered twice with Miracloth (Millipore, cat # 475855), the filtrate was collected, centrifuged at 4 ℃ for 15min at 3000g, 37% by volume of aqueous formaldehyde was added to the supernatant obtained by centrifugation in an amount of 800. mu.L per 2g of cotton callus (so that the final concentration of formaldehyde in the system was 1% by volume), fixed by crosslinking at room temperature for 8min, and terminated by addition of glycine (solvent is water) at a concentration of 2.5M.
5. Cell nuclei were recovered by centrifugation at 1500g and 4 ℃ for 5 min.
Conventional methods：
1. Collecting 4g to 10g of fresh tissue sample;
2. add 45mL PBS (or sugarless MS) and 1.25mL 37% formaldehyde solution (sigma) in a fume hood (final concentration 1%) to each 50mL centrifuge tube;
3. each 2-3g sample was cut into 2 mm. times.3 mm. times.10 mm pieces with a sharp blade, placed in the above 50mL centrifuge tube, evacuated at room temperature to-60 to-80 Kpa, kept for 10min, and then returned to atmospheric pressure. Repeating the above steps for three times (standard: sample sinking);
4. the crosslinking reaction was terminated by adding 2.5mL of 2.5M glycine (stored at 4 ℃ C.) (final concentration: 0.125M), followed by shaking for 5min to terminate the reaction.
5. Filtrate off the liquid, ddH2Cleaning for 3 times, drying the liquid on the surface of the sample by using absorbent paper, quickly freezing by using liquid nitrogen, storing at-80 ℃, and transporting by using dry ice.
6. The crosslinked sample was ground well into a powder with liquid nitrogen and transferred to a 50mL centrifuge tube containing 10mL ice-chilled NIbuffer (N)Ibuffer，20mM Hepes pH 8,250mM Sucrose,1mM MgCl25mM KCl, 40% (v/v) glycerol, 0.25% (v/v) Triton X-100,0.1mM PMSF, 0.1% (v/v) β -ME,1/5cocktail), gently shaken on ice for 15 min.
7. The suspension was filtered twice through miracloth (Millipore), the filtrate collected in a 50mL centrifuge tube and centrifuged at 3000g for 15min at 4 ℃.
(II) library building process
1. The supernatant was discarded and the pellet was resuspended in 2mL of NIbuffer and centrifuged at 1900g for 5min at 4 ℃.
2. Repeat step 1 once.
3. The supernatant was discarded. Resuspend in 1mL of 1.2 XNEBuffer 2, centrifuge at 1900g for 5min at 4 ℃.
4. The supernatant was discarded and resuspended in 500. mu.L of 1.2 XNEBuffer 2.
5. 7.5 μ L of 20% (w/v) SDS was added. Incubate at 65 ℃ for 40 min. Incubate at 37 ℃ for 20min with a shaker at 800 rpm.
6. 50 μ L of 20% (v/v) Triton X-100 was added and incubated at 37 ℃ for 1h with a shaker at 800 rpm.
7. Add 50. mu.L NEB buffer3.1, 10. mu.L DpnII and spin overnight at 37 ℃.
8. DpnII was inactivated at 62 ℃ for 20 minutes.
9. Biotin-labeled DNA (1.5. mu.L 10mM dATP, 1.5. mu.L 10mM dGTP, 1.5. mu.L 10mM dTTP, 37.5. mu.L 0.4mM biotin-14-dCTP) and 10. mu.L 5U/. mu.L Klenow enzyme-filled DNA were added, carefully mixed and incubated at 37 ℃ for 45 min.
10. The reaction mixture was removed and placed on ice, and 86. mu.L of 10% (w/v) SDS was added to terminate the reaction. Incubate at 65 ℃ for 30min, then quickly place on ice.
11. The DNA was filled in, and 7.61mL of a loop reaction system (745. mu.L of 10% (v/v) Triton X-100,745. mu.L of 10 × ligation buffer (500mM Tris-HCl pH 7.5,100mM MgCl) was added to ice2100mM DTT), 80. mu.L of 10mg/mL BSA, 80. mu.L of 100mM ATP and 5.96mL ddH2O) to a 15mL centrifuge tube, transfer chromatin to the 15mL centrifuge tube.
12. 50U T4 DNA ligase was added thereto, the mixture was inverted and mixed, and reacted at 16 ℃ for 4 hours to form a loop of DNA.
13. Add 50. mu.L 10mg/mL proteinase K and digest overnight at 65 ℃. Then, 50. mu.L of 10mg/mL proteinase K was added and digested at 65 ℃ for 2 hours.
14. Cooled to room temperature and transferred to a 50mL centrifuge tube. DNA was purified by phenol extraction. Add 10mL phenol pH8.0 and vortex for 2 min. Centrifuge at 1500g for 10min and transfer the aqueous phase to a new 50mL centrifuge tube.
15. Re-extraction and purification by phenol chloroform (1: 1 volume), vortexing and shaking for 2min, centrifuging at 22000g for 5min, and transferring the aqueous phase to a new 50mL centrifuge tube.
16. Adding 2.5 times volume of pure alcohol to precipitate DNA, centrifuging at 22000g for 5min, and discarding the supernatant.
17. The DNA pellet was dissolved by adding 450. mu.L of TE (pH 8.0) and transferred to a 2mL centrifuge tube.
18. Add 500. mu.L phenol chloroform (1: 1 vol), shake for 1min, centrifuge at 22000g for 5min, transfer the aqueous phase to a new tube. After two purifications, 0.1 volume of NaOAc and 2 volumes of absolute alcohol were added, incubated at-80 ℃ for 30min and the supernatant was centrifuged off.
19. The DNA was washed by adding 1mL of 70% ethanol and centrifuged at 22000g for 5 min. The supernatant was discarded.
20. Add 25. mu.L of TE to resuspend the DNA. Add 1. mu.L of 1mg/mL RNase A and digest for 30min at 37 ℃.
21. Quality inspection: a5. mu.L sample was taken for detection on 1% agarose gel. And the sample with obvious main band is unqualified and needs to be treated and then is subjected to enzyme digestion. If a diffuse band is present, the sample continues to follow-up.
22. The unclycled biotin-labeled DNA was removed using the exonuclease activity of T4 DNA polymerase. Mu.g of library was added to 100. mu.L of a system containing 1. mu.L of 10mg/mL BSA, 10. mu.L of 10 XNEBuffer 2, 1. mu.L of 10mM dATP, 1. mu.L of 10mM dGTP and 5 Units T4 DNA polymerase, and incubated at 16 ℃ for 4 hours.
23. The reaction was stopped by adding 2. mu.L of 0.5M EDTA, pH8.0, and the DNA was purified by phenol chloroform (1: 1, volume ratio)/ethanol precipitation. 100 μ L ddH2O resuspend DNA.
24. The DNA was disrupted to around 200-700bp using an ultrasonic DNA disruptor (Covaris M220).
25. The ends were repaired by adding 14. mu.L of 10 × ligation buffer, 14. mu.L of 2.5mM dNTPmix, 5. mu. L T4 DNApolymerase, 5. mu. L T4 nucleotide kinase, 1. mu.L of Klenow DNA polymerase and 1. mu.L of water and incubating at 20 ℃ for 30 min.
26. DNA purification was performed by DNA purification kit (Qiagen). After elution twice with 15. mu.L of low-salt TE (TLE:10mM Tris pH8.0,0.1mM EDTA), the DNA was added with A and incubated for 20 minutes at 37 ℃ with 5. mu.L of 10 XNEBuffer 2, 10. mu.L of 1mM dATP, 2. mu.L of water and 3. mu.L of Klenow (exo-).
27. The reaction was stopped by incubation at 65 ℃ for 20min and quickly placed on ice. Concentrate to 20 μ L in vacuo.
28. The DNA was recovered by running on a 1.5% agarose gel 80-90v for 3.5 hours. The DNA of 300-500bp is selected, cut and recovered by a gel recovery kit. Elute with 50 μ L TLE. The eluates were pooled to 300. mu.L. The total amount of DNA was calculated by Qubit.
29. The following experiments are recommended to be performed in low DNA adsorption tubes (DNA Lobind tubes).
30. Biotin DNA enrichment was performed by the magnetic bead method, with detailed reference to kit instructions (AgencourtAmpure areas, Beckman). Specifically, the method comprises the following steps:
A. biotin pull-down beads were prepared by washing 150. mu.L of resuspended streptavidin magnetic beads twice with 400. mu.L of LTween buffer (TB: 5mM Tris-HCl pH8.0, 0.5mM EDTA, 1M NaCl, 0.05% Tween).
B. The magnetic beads were resuspended in 300. mu.L of 2X buffer without Tween (2 XTB: 10mM Tris-HCl pH8.0, 1mM EDTA, 2M NaCl) and 300. mu. LHi-C DNA obtained in step 28 was added. The mixture was incubated at room temperature for 15 minutes while rotating, and then biotin-labeled Hi-C DNA was bound to streptavidin-containing magnetic beads.
C. Streptavidin magnetic beads bound to DNA were recovered with a magnetic particle concentrator and the supernatant was discarded. The beads were washed sequentially with 400. mu.L of 1 XNTB (5mM Tris-HCl pH8.0, 0.5mM EDTA, 1M NaCl) and 100. mu.L of 1 Xligation buffer. The beads were then resuspended in 50. mu.L of 1 × ligation buffer, and the mixture was transferred to a new tube.
D. The magnetic beads were resuspended in Gibson kit buffer and digested for 10min at 37 ℃ with T5 exonuclease (ref: enzymatic assembly of DNA molecules up to sectional cloned kits).
E. The ends were repaired by adding 14. mu.l of 10 Xligation buffer, 14. mu.l of 2.5mM dNTPmix, 5. mu. l T4 DNApolymerase, 5. mu. l T4 nucleotide kinase, 1. mu.l Klenow DNA polymerase and 1. mu.l water and incubating at 20 ℃ for 30 min.
F. DNA purification was performed by DNA purification kit (Qiagen). After elution twice with 15. mu.l of low-salt TE (TLE:10mM Tris pH8.0,0.1mM EDTA), the DNA was added with A and incubated for 20 minutes at 37 ℃ with 5. mu.l of 10 XNEBuffer 2, 10. mu.l of 1mM dATP, 2. mu.l of water and 3. mu.l of Klenow (exo-).
The reaction was stopped by incubation at G.65 ℃ for 20min and quickly placed on ice.
H. The total amount of DNA calculated in step 28 was used as the input for biotin pulldown and divided by 20 to estimate the total amount of Hi-C DNA that was pulled down and available for linker ligation and sequencing. The adaptors were added at a rate of 6 picomoles of Illumina double-ended linker per microgram of Hi-C DNA available for ligation. 1200 units of T4 DNA ligase was used to ligate the linker to the DNA. Incubate at room temperature for 2 hours.
I. The magnetic beads bound to the Hi-C DNA were recovered and washed twice with 400. mu.L of 1 XTB, thereby removing the excess double-ended linker.
J. The beads were washed sequentially with 200. mu.L of 1 XNTB, 200. mu.L and 50. mu.L of 1 XNEBuffer. After the last wash, it was resuspended in 50. mu.L of 1 XNEBuffer 2 and transferred to a new tube.
K. To determine the appropriate number of cycles to generate enough PCR products to be sequenced, PCR experiments were performed at 6, 9, 12 and 15 cycles, respectively. The optimal number of cycles was determined by PCR reactions on a 5% polyacrylamide gel and staining with Sybr Green, ensuring that no bands were present and that bands appeared in the 400-base pair segment, since this is the approximate fragment length after ligation of the Hi-C DNA to the linker.
L. Large scale PCR was performed at the optimal number of PCR cycles to amplify the remaining Hi-C DNA library bound to magnetic beads. The PCR products were collected separately and the magnetic beads were recovered. 1% of the PCR product was placed on a gel as a control, and the remaining PCR product was purified using 1.8 volumes of Ampure beads.
M. eluting the purified PCR product by 50 mu L of 1 XTLE buffer solution, namely the constructed Hi-C library.
Through the above-described procedures, three Hi-C libraries obtained by the FS-Hi-C method of the present invention (designated as CF9, CF10, and CF15) were obtained, and three Hi-C libraries obtained by the conventional method (designated as MH14, MH16, and MH19) were obtained.
(III) Hi-C high throughput sequencing
Pair-end sequencing (paired-end sequencing) was performed using an Illumina: HiSeq X Ten sequencer.
(IV) quality identification of Hi-C library
The high throughput sequencing data of 6 Hi-C libraries obtained by the conventional and inventive methods were evaluated. The data quality analysis was performed using the high-efficiency Hi-C data preprocessing tool HiC-Pro, and the quality control results are shown in Table 2. The result shows that the ratio of the uniquely matched double-ended sequencing reads obtained by the method is stable, the ratio of the interactive double-ended reads is high, the library capacity is increased along with the increase of the sequencing depth, the effective data is increased, and the ratio of invalid data caused by PCR amplification is low and is maintained at 21-25%. In contrast, the traditional method results in a reduced proportion of interactive double-ended reads, a smaller library size of the constructed library, and a larger proportion of invalid data for PCR amplification (67-73%).
And (4) conclusion: the quality of the Hi-C library of the method is superior to that of the library built by the traditional method.
TABLE 2 quality control results of Hi-C library high throughput sequencing data of the present and conventional methods generated by the software HiCPro
Figure BDA0002532603870000131
Compared with the traditional Hi-C method, the Hi-Cmap between the same chromosomes of the frozen replacement Hi-C (FS-Hi-C) shows high similarity (FIG. 5); compared with the traditional Hi-C method, the distribution pattern of A and B components between the same chromosome is similar (FIG. 6); compared with the conventional Hi-C method, the freezing replacement Hi-C (FS-Hi-C) of the invention has the advantages that the distribution pattern of TAD detected on the same chromosome is similar (FIG. 7); chromatin structure was highly similar for the frozen replacement (FS) and fresh samples of the invention (fig. 8). These results demonstrate that at the genome-wide level, the three-dimensional conformation of chromatin exhibits a high degree of similarity between frozen replacement of Hi-C (FS-Hi-C) and the traditional Hi-C method, and FS-Hi-C exhibits a higher library quality.
In addition, the invention also adopts freezing to replace Hi-C (FS-Hi-C) and traditional methods to respectively carry out Hi-C library construction, Hi-C high-throughput sequencing and result analysis on the tissue samples of the soybeans and the radishes. As shown in Table 3, in soybean and radish, the method of the present invention produced an increased proportion of paired ends matched to unique sites in the genome and a decreased proportion of repetitive sequences produced by PCR amplification compared to the conventional method.
And (4) conclusion: the quality of the freezing replacement Hi-C (FS-Hi-C) library is superior to that of the library built by the traditional method, and the method detects more effective reads under the same raw reads.
TABLE 3 the rapid freeze-crosslinking technique of the present invention can effectively increase the effective data volume of the library
Figure BDA0002532603870000141
The results of the embodiments of the invention are combined, and it can be seen that: the Hi-C high-throughput sequencing library construction method provided by the invention proves that rapid freezing and then crosslinking do not change chromatin conformation of species by improving a pretreatment mode, improves the application range of the Hi-C technology, and can solve the problem of insufficient crosslinking caused by the existence of plant cell walls and vacuoles.
Example 2 Hi-C high-throughput sequencing of Drosophila
The present example uses Drosophila as the subject of study.
First, sample pretreatment
The method of the invention：
1. Collecting Drosophila cell line (S2 cell line) 5 × 106Cells were snap frozen in liquid nitrogen and ground to a powder.
2. Transferring the ground substance to a pre-frozen cross-linking solution at-90 ℃, and then slowly raising the temperature in a gradient way: 6h at 90 ℃ below zero; the temperature is between 90 ℃ below zero and 60 ℃ below zero for 6 hours (the temperature is raised by 5 ℃ per hour); 6h at 60 ℃ below zero; the temperature is between 60 ℃ below zero and 30 ℃ below zero for 6 hours (the temperature is raised by 5 ℃ per hour); 6h at-30 ℃; -30 ℃ to 0 ℃ for 6h (5 ℃ per hour); each temperature gradient lasted 6 h. The water in the cells is replaced by an ethanol solution.
3. Centrifuging at 4 deg.C for 30s at 3000g, removing ethanol solution, washing with NIbuffer solution pre-cooled on ice for three times, and washing at 2 × 10 times620mL of each cell was added to a pre-chilled (pre-chilled on ice) NIbuffer to form a suspension and gently shaken for 15 min.
4. The suspension was filtered twice with Miracloth (Millipore, cat # 475855), the filtrate was collected, centrifuged at 4 ℃ at 3000g for 15min, and the supernatant from the centrifugation was centrifuged at 2 × 106An amount of 800. mu.L per cell was added 37% by volume of formaldehyde in water (so that the final concentration of formaldehyde in the system was 1% by volume), fixed for 8min at room temperature, and terminated by the addition of 2.5M glycine (the solvent was water).
5. Cell nuclei were recovered by centrifugation at 1500g and 4 ℃ for 5 min.
Conventional methods：
1. Collecting Drosophila cell line (S2 cell line), and collecting 5 × 106(ii) individual cells;
2. add 45mL PBS (or sugarless MS) and 1.25mL 37% formaldehyde solution (sigma) in a fume hood (final concentration 1%) to each 50mL centrifuge tube;
3. the sample was placed in the above 50mL centrifuge tube, evacuated at room temperature from-60 to-80 Kpa, kept for 10min, and then returned to atmospheric pressure. Repeating the above steps for three times (standard: sample sinking);
4. the crosslinking reaction was terminated by adding 2.5mL of 2.5M glycine (stored at 4 ℃ C.) (final concentration: 0.125M), followed by shaking for 5min to terminate the reaction.
5. Filtrate off the liquid, ddH2Cleaning for 3 times, drying the liquid on the surface of the sample by using absorbent paper, quickly freezing by using liquid nitrogen, storing at-80 ℃, and transporting by using dry ice.
6. After crosslinking, the sample was ground well into a powder with liquid nitrogen and transferred to a 50mL centrifuge tube containing 10mL ice-chilled NIbuffer (NIbuffer, 20mM Hepes pH8, 250mM Cross, 1mM MgCl)2,5mM KCl,40％(v/v)glycerol, 0.25％(v/v) Triton X-100,0.1mM PMSF, 0.1% (v/v) β -ME,1/5cocktail), gently shaken on ice for 15 min.
7. The suspension was filtered twice through miracloth (Millipore), the filtrate collected in a 50mL centrifuge tube and centrifuged at 3000g for 15min at 4 ℃.
Second, building a library process
1. The supernatant was discarded and the pellet was resuspended in 2mL of NIbuffer and centrifuged at 1900g for 5min at 4 ℃.
2. Repeat step 1 once.
3. The supernatant was discarded. Resuspend in 1mL of 1.2 XNEBuffer 2, centrifuge at 1900g for 5min at 4 ℃.
4. The supernatant was discarded and resuspended in 500. mu.L of 1.2 XNEBuffer 2.
5. 7.5 μ L of 20% (w/v) SDS was added. Incubate at 65 ℃ for 40 min. Incubate at 37 ℃ for 20min with a shaker at 800 rpm.
6. 50 μ L of 20% (v/v) Triton X-100 was added and incubated at 37 ℃ for 1h with a shaker at 800 rpm.
7. Add 50. mu.L NEB buffer3.1, 10. mu.L DpnII and spin overnight at 37 ℃.
8. DpnII was inactivated at 62 ℃ for 20 minutes.
9. Biotin-labeled DNA (1.5. mu.L of 10mM dATP, 1.5. mu.L of 10mM dGTP, 1.5. mu.L of 10mM dTTP, 37.5. mu.L of 0.4mM biotin-14-dCTP) and 10. mu.L of 5U/. mu.L Klenow enzyme-filled DNA were added, carefully mixed and incubated at 37 ℃ for 45 min.
10. The reaction mixture was removed and placed on ice, and 86. mu.L of 10% (w/v) SDS was added to terminate the reaction. Incubate at 65 ℃ for 30min, then quickly place on ice.
11. The DNA was filled in, and 7.61mL of a loop reaction system (745. mu.L of 10% (v/v) Triton X-100,745. mu.L of 10 × ligation buffer (500mM Tris-HCl pH 7.5,100mM MgCl) was added to ice2100mM DTT), 80. mu.L of 10mg/mL BSA, 80. mu.L of 100mM ATP and 5.96mL ddH2O) to a 15mL centrifuge tube, transfer chromatin to the 15mL centrifuge tube.
12. 50U T4 DNA ligase was added thereto, the mixture was inverted and mixed, and reacted at 16 ℃ for 4 hours to form a loop of DNA.
13. Add 50. mu.L 10mg/mL proteinase K and digest overnight at 65 ℃. Then, 50. mu.L of 10mg/mL proteinase K was added and digested at 65 ℃ for 2 h.
14. Cooled to room temperature and transferred to a 50mL centrifuge tube. DNA was purified by phenol extraction. Add 10mL phenol pH8.0 and vortex for 2 min. Centrifuge at 1500g for 10min and transfer the aqueous phase to a new 50mL centrifuge tube.
15. Re-extraction and purification by phenol chloroform (1: 1 volume), vortexing and shaking for 2min, centrifuging at 22000g for 5min, and transferring the aqueous phase to a new 50mL centrifuge tube.
16. 2.5 times volume of absolute ethanol is added to precipitate DNA, 22000g is centrifuged for 5min, and the supernatant is discarded.
17. The DNA pellet was dissolved by adding 450. mu.L of TE (pH 8.0) and transferred to a 2mL centrifuge tube.
18. Add 500. mu.L phenol chloroform (1: 1 vol), shake for 1min, centrifuge at 22000g for 5min, transfer the aqueous phase to a new tube. After two purifications, 0.1 volumes of NaOAc and 2 volumes of absolute ethanol were added, incubated at-80 ℃ for 30min and the supernatant centrifuged off.
19. The DNA was washed by adding 1mL of 70% ethanol and centrifuged at 22000g for 5 min. The supernatant was discarded.
20. Add 25. mu.L of TE to resuspend the DNA. Add 1. mu.L of 1mg/mL RNase A and digest for 30min at 37 ℃.
21, quality inspection: a5. mu.L sample was taken for detection on 1% agarose gel. And the sample with obvious main band is unqualified and needs to be treated and then is subjected to enzyme digestion. If a diffuse band is present, the sample continues to follow-up.
22. The unclycled biotin-labeled DNA was removed using the exonuclease activity of T4 DNA polymerase. Mu.g of library was added to 100. mu.L of a system containing 1. mu.L of 10mg/mL BSA, 10. mu.L of 10 XNEBuffer 2, 1. mu.L of 10mM dATP, 1. mu.L of 10mM dGTP and 5 Units T4 DNA polymerase, and incubated at 16 ℃ for 4 hours.
23. The reaction was stopped by adding 2. mu.L of 0.5M EDTA, pH8.0, and the DNA was purified by phenol chloroform (1: 1, volume ratio)/ethanol precipitation. 100 μ L ddH2O resuspend DNA.
24. The DNA was disrupted to around 200-700bp using an ultrasonic DNA disruptor (Covaris M220).
25. The ends were repaired by adding 14. mu.L of 10 × ligation buffer, 14. mu.L of 2.5mM dNTPmix, 5. mu. L T4 DNApolymerase, 5. mu. L T4 nucleotide kinase, 1. mu.L of Klenow DNA polymerase and 1. mu.L of water and incubating at 20 ℃ for 30 min.
26. DNA purification was performed by DNA purification kit (Qiagen). After elution twice with 15. mu.L of low-salt TE (TLE:10mM Tris pH8.0,0.1mM EDTA), the DNA was added with A and incubated for 20 minutes at 37 ℃ with 5. mu.L of 10 XNEBuffer 2, 10. mu.L of 1mM dATP, 2. mu.L of water and 3. mu.L of Klenow (exo-).
27. The reaction was stopped by incubation at 65 ℃ for 20min and quickly placed on ice. Concentrate to 20 μ L in vacuo.
28. The DNA was recovered by running on a 1.5% agarose gel 80-90v for 3.5 hours. The DNA of 300-500bp is selected, cut and recovered by a gel recovery kit. Elute with 50 μ L TLE. The eluates were pooled to 300. mu.L. The total amount of DNA was calculated by Qubit.
29. The following experiments are recommended to be performed in low DNA adsorption tubes (DNA Lobind tubes).
30. Biotin DNA enrichment was performed by the magnetic bead method, with detailed reference to kit instructions (AgencourtAmpure areas, Beckman). Specifically, the method comprises the following steps:
A. biotin pull-down beads were prepared by washing 150. mu.L of resuspended streptavidin magnetic beads twice with 400. mu.L of LTween buffer (TB: 5mM Tris-HCl pH8.0, 0.5mM EDTA, 1M NaCl, 0.05% Tween).
B. The magnetic beads were resuspended in 300. mu.L of 2X buffer without Tween (2 XTB: 10mM Tris-HCl pH8.0, 1mM EDTA, 2M NaCl) and 300. mu. LHi-C DNA obtained in step 28 was added. The mixture was incubated at room temperature for 15 minutes while rotating, and then biotin-labeled Hi-C DNA was bound to streptavidin-containing magnetic beads.
C. Streptavidin magnetic beads bound to DNA were recovered with a magnetic particle concentrator and the supernatant was discarded. The beads were washed sequentially with 400. mu.L of 1 XNTB (5mM Tris-HCl pH8.0, 0.5mM EDTA, 1M NaCl) and 100. mu.L of 1 Xligation buffer. The beads were then resuspended in 50. mu.L of 1 × ligation buffer, and the mixture was transferred to a new tube.
D. The magnetic beads were resuspended in Gibson kit buffer and digested for 10min at 37 ℃ with T5 exonuclease (ref: enzymatic assembly of DNA molecules up to sectional cloned kits).
E. The ends were repaired by adding 14. mu.l of 10 Xligation buffer, 14. mu.l of 2.5mM dNTPmix, 5. mu. l T4 DNApolymerase, 5. mu. l T4 nucleotide kinase, 1. mu.l Klenow DNA polymerase and 1. mu.l water and incubating at 20 ℃ for 30 min.
F. DNA purification was performed by DNA purification kit (Qiagen). After elution twice with 15. mu.l of low-salt TE (TLE:10mM Tris pH8.0,0.1mM EDTA), the DNA was added with A and incubated for 20 minutes at 37 ℃ with 5. mu.l of 10 XNEBuffer 2, 10. mu.l of 1mM dATP, 2. mu.l of water and 3. mu.l of Klenow (exo-).
The reaction was stopped by incubation at G.65 ℃ for 20min and quickly placed on ice.
H. The total amount of DNA calculated in step 28 was used as the input for biotin pulldown and divided by 20 to estimate the total amount of Hi-C DNA that was pulled down and available for linker ligation and sequencing. The adaptors were added at a rate of 6 picomoles of Illumina double-ended linker per microgram of Hi-C DNA available for ligation. 1200 units of T4 DNA ligase was used to ligate the linker to the DNA. Incubate at room temperature for 2 hours.
I. The magnetic beads bound to the Hi-C DNA were recovered and washed twice with 400. mu.L of 1 XTB, thereby removing the excess double-ended linker.
J. The beads were washed sequentially with 200. mu.L of 1 XNTB, 200. mu.L and 50. mu.L of 1 XNEBuffer. After the last wash, it was resuspended in 50. mu.L of 1 XNEBuffer 2 and transferred to a new tube.
K. To determine the appropriate number of cycles to generate enough PCR products to be sequenced, PCR experiments were performed at 6, 9, 12 and 15 cycles, respectively. The optimal number of cycles was determined by PCR reactions on a 5% polyacrylamide gel and staining with Sybr Green, ensuring that no bands were present and that bands appeared in the 400-base pair segment, since this is the approximate fragment length after ligation of the Hi-C DNA to the linker.
L. Large scale PCR was performed at the optimal number of PCR cycles to amplify the remaining Hi-C DNA library bound to magnetic beads. The PCR products were collected separately and the magnetic beads were recovered. 1% of the PCR product was placed on a gel as a control, and the remaining PCR product was purified using 1.8 volumes of Ampure beads.
M. eluting the purified PCR product by 50 mu L of 1 XTLE buffer solution, namely the constructed Hi-C library.
Through the above-mentioned procedures, two Hi-C libraries (designated as S2-1 and S2-5) obtained by the method of the present invention and two Hi-C libraries (designated as SRR7187330 and SRR7187332) obtained by the conventional method were obtained.
Three, Hi-C high throughput sequencing
Pair-end sequencing (paired-end sequencing) was performed using an Illumina: HiSeq X Ten sequencer.
Quality identification of four, Hi-C library
The high throughput sequencing data of 4 Hi-C libraries obtained by the present invention, frozen replacement of Hi-C (FS-Hi-C) and the conventional method, were evaluated (S2-1 and S2-5 are libraries constructed by the present invention, and SRR7187330 and SRR7187332 are libraries constructed by the conventional method). The data quality analysis was performed using the high-efficiency Hi-C data preprocessing tool HiC-Pro, and the quality control results are shown in Table 4. The results show that compared with the traditional method, the number of the credible double-ended sequencing fragments obtained by the method, the number and the ratio of the double-ended sequencing fragments compared to the unique locus of the genome, the number of the double-ended sequencing fragments with interaction and the number of the double-ended sequencing fragments with effective interaction are all obviously improved.
And (4) conclusion: the quality of the Hi-C library of the method is superior to that of the library built by the traditional method.
TABLE 4 quality control results of high throughput sequencing data of Hi-C libraries of the present and conventional methods generated by software HiC-Pro
Figure BDA0002532603870000181
The Hi-C maps of 2L, 2R, 3L, 3R, 4 and X chromosomes all show a high degree of similarity compared to the conventional Hi-C method for the cryo-replacement of Hi-C (FS-Hi-C) of the present invention. The library quality of FS-Hi-C was high, the effective data was high, and the frequency of detected DNA interactions was increased (FIG. 9). In the case of raw reads being close, the A and B component are distributed similarly overall compared to the Hi-C method (FIG. 10). FS-Hi-C detected a similar overall distribution of TAD in the same chromosome compared to the traditional Hi-C method (FIG. 11). These results indicate that the FS-Hi-C method does not affect the three-dimensional conformation of Drosophila chromatin.
Combining the results of the two examples of the present invention, it can be seen that: the invention provides a method for constructing a library of a freezing replacement Hi-C (FS-Hi-C) suitable for organisms. The research breaks through the limitation that the Hi-C treatment needs to carry out formaldehyde fixation on a fresh sample and then cross-linking, can store the sample in liquid nitrogen for later use, and provides an improved method for the application of Hi-C in organisms.

Claims (10)

1. A sample pretreatment method suitable for Hi-C high-throughput sequencing and library building comprises the following steps: the biological sample to be tested is frozen and then cross-linked and fixed.
2. The method of claim 1, wherein: the freezing is low-temperature treatment at minus 196 ℃;
further, the freezing is that the biological sample to be tested is put into liquid nitrogen for quick freezing;
and/or
The cross-linking fixation is carried out by using formaldehyde solution;
and/or
And the process of pre-freezing and crosslinking the biological sample to be tested and gradient temperature rise is also included between the freezing and the crosslinking and fixing.
3. The method according to claim 1 or 2, characterized in that: the method comprises the following steps of sequentially processing the biological sample to be tested:
(A1) quick-freezing and grinding by using liquid nitrogen;
(A2) pre-freezing and crosslinking;
(A3) gradient heating;
(A4) centrifuging, adding NIbuffer and filtering;
(A5) formaldehyde crosslinking and fixing;
(A6) glycine terminates crosslinking;
(A7) the cell nuclei were recovered by centrifugation.
4. The method of claim 3, wherein: in the step (a1), the test biological sample is put into liquid nitrogen for quick freezing and ground into powder in the liquid nitrogen;
and/or
The pre-frozen cross-linking of claim 2 is carried out in a pre-frozen cross-linking solution comprising 2% water, 0.01% formaldehyde in an alcohol solution,% by volume;
and/or
In step (A2) of claim 3, transferring the (A1) milled powder to the pre-frozen cross-linking liquid at-90 ℃;
and/or
The gradient temperature rise is 6h at-90 ℃, 6h at-60 ℃, 6h at-30 ℃ and 6h at 0 ℃;
further, in the gradient temperature rise process, the temperature rises from-90 ℃ to-60 ℃ by 5 ℃ per hour, the temperature rises from-60 ℃ to-30 ℃ by 5 ℃ per hour, and the temperature rises from-30 ℃ to 0 ℃ by 5 ℃ per hour;
and/or
In step (a4), the recipe for NIbuffer is as follows: 20mM Hepes pH8, 250mM sucrose,1mM magnesium chloride, 5mM potassium chloride, 40% glycerol by volume, 0.25% Triton X-100 by volume, 0.1mM PMSF, 0.1% beta-mercaptoethanol by volume, 1/5 volume fraction of cocktail;
and/or
In step (a4), removing the supernatant after the centrifugation, adding the NIbuffer suspension precipitate pre-cooled, filtering with Miracloth, collecting the filtrate, and centrifuging;
and/or
The formaldehyde solution of claim 2 which is 37% by volume formaldehyde solution;
and/or
The method of claim 3, wherein in step (A5), the supernatant from the centrifugation in step (A4) is added with 37% by volume of aqueous formaldehyde solution for crosslinking fixation, wherein the crosslinking temperature is room temperature and the crosslinking time is 8 min;
and/or
When the crosslinking and fixing are carried out, the final concentration of the formaldehyde in the system is 1 percent by volume.
5. The method of claim 4, wherein: in step (A6), the crosslinking was terminated by adding a 2.5M glycine solution.
6. A Hi-C high-throughput sequencing and database building method comprises the following steps: pre-treating a test biological sample by the method of any one of claims 1 to 5; the treated samples were then subjected to Hi-C high throughput sequencing for pooling.
7. A Hi-C high-throughput sequencing method comprises the following steps: pre-treating a test biological sample by the method of any one of claims 1 to 5; then carrying out Hi-C high-throughput sequencing on the treated sample to build a library; and finally performing Hi-C high-throughput sequencing.
8. The method according to any one of claims 1-7, wherein: the biological sample is a cell or tissue.
9. The method according to any one of claims 1-8, wherein: the organism is a plant or an animal;
further, the plant is cotton or soybean or radish; the animal is a fruit fly;
further, the biological sample is callus of cotton or a drosophila cell line.
10. Use in any of the following:
(B1) use of the method of any one of claims 1-5 in Hi-C high throughput sequencing pooling;
(B2) use of the method of any one of claims 1-6 in Hi-C high throughput sequencing.
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