Positioning and quantifying method for efficiently obtaining whole genome of nucleosome on metaphase chromosome

Abstract

The invention discloses a positioning and quantifying method for efficiently acquiring a whole genome of a nucleosome on a metaphase chromosome, which comprises the following steps: preparing and enriching metaphase cells, and purifying the metaphase chromosome by a centrifugal filter membrane integrated method; resuspending the metaphase chromosome precipitate by adopting an optimized MNase buffer, protecting a metaphase chromosome structure, and keeping the position of a nucleosome unchanged; adding MNase enzyme for enzyme digestion reaction at low temperature (4 DEG C); adding EDTA (Ethylene Diamine Tetraacetic Acid) to terminate enzyme digestion reaction to obtain metaphase chromosome monokaryosome suspension; dNA (deoxyribonucleic acid) extraction and quantification; a quantifiable UMI joint is combined with a DNA library construction kit to carry out library construction, and sequencing and analysis are carried out after quality detection. According to the method, the mononuclear bodies on the metaphase cell chromosomes can be efficiently and stably obtained, the whole genome of the mononuclear bodies on the metaphase chromosomes can be positioned and quantified by combining quantitative library building and high-throughput sequencing technologies, the result is stable, and the quantification is accurate.

Description

Technical Field

[0001] The invention belongs to the field of biotechnology, and in particular relates to a method for efficiently obtaining the positioning and quantification of the whole genome of nucleosomes on metaphase chromosomes. Background Art

[0002] Nucleosomes are the most basic structural and regulatory units of chromatin and chromosomes, and the higher-order structures of chromosomes and chromatin are formed through the folding and condensation of these nucleosomes. Most of the sequences of genomic DNA are packaged on nucleosomes, and the occupancy and distribution of nucleosomes not only directly affect the regulation of gene expression, but also restrict the morphology of higher-order structures. Therefore, it is of great significance to explore the distribution of nucleosomes on the genome.

[0003] At present, there have been a lot of studies on the distribution of nucleosomes in interphase cells on the genome, as well as various variants or modifications of histones, but due to technical limitations, there are few studies on the distribution of metaphase chromosomes. Micrococcal nuclease (MNase) is a nuclease that can degrade DNA in the nucleosome junction region. The enzyme has been used to prepare mononucleosomes in interphase chromatin, but the unique high condensation structure of metaphase chromosomes makes the conventional MNase enzyme digestion method for preparing mononucleosomes unable to be applied to this field. Therefore, it is necessary to establish a suitable method to enable it to be applied to metaphase chromosomes, and it is necessary to meet the following requirements at the same time to ensure that the nucleosome positioning under the physiological state of the whole genome is obtained: first, to ensure that the distribution state of nucleosomes under the enzyme digestion system is consistent with that under physiological conditions; second, to ensure that the enzyme digestion preparation process does not affect the position of nucleosomes, so as to truly obtain the positioning information of the original nucleosomes; third, the chromosome must be efficiently and fully sheared into single nucleosomes to obtain the information of nucleosomes in the whole genome; fourth, to design a suitable UMI sequence for DNA library construction to obtain more accurate high-throughput sequencing data, so as to quantitatively analyze the nucleosome distribution.

[0004] At present, conventional methods for preparing mononucleosomes use MNase digestion technology, which is designed for interphase cells after permeabilization. Therefore, there is still a lack of an efficient method for obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes. Summary of the invention

[0005] In view of the above-mentioned deficiencies in the prior art, the purpose of the present invention is to provide a method for efficiently obtaining the positioning and quantification of the whole genome of nucleosomes on metaphase chromosomes, which is combined with DNA library construction using UMI adapters to effectively obtain more accurate quantitative data. The steps are simple and can be widely used for the positioning and accurate quantification of nucleosomes on metaphase chromosomes of various samples.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows:

[0007] A method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes, comprising the following steps:

[0008] S1. Prepare and enrich metaphase cells, and purify metaphase chromosomes by centrifugal filter integration;

[0009] S2. Use the independently developed and optimized MNase buffer to resuspend the metaphase chromosome pellet, protect the metaphase chromosome structure, and keep the nucleosome position unchanged;

[0010] S3, add MNase enzyme with a final concentration of 1000-3,000 gel units / mL and a chromosome concentration of 100-1000 ng, and perform the enzyme digestion reaction at 4°C for 12-24 hours to achieve uniform enzyme digestion of metaphase chromosomes;

[0011] S4, adding EDTA at a final concentration of 20 mM to terminate the enzyme digestion reaction and obtain a mononucleosome suspension of metaphase chromosomes;

[0012] S5. Use a DNA extraction kit to extract and quantify DNA;

[0013] S6. The initial adapter is connected to the index containing random hexamer to obtain the UMI adapter, and the library is constructed by combining with the DNA library construction kit. The obtained library is sequenced and analyzed after passing the quality test.

[0014] Preferably, in step S1, the membrane of the enriched metaphase cells is broken by a homogenizer, the obtained homogenate is filtered through 10 μm and 5 μm filter membranes, centrifuged, and the precipitate is collected to obtain pure metaphase chromosomes.

[0015] More preferably, in step S1, the centrifugation conditions are 4°C, 1750g, and 10 min.

[0016] Preferably, in step S2, the formula of the MNase buffer is: 50mM Tris-HCl, 0.2mMspermine, 0.5mM spermidine, 80mM KCl, 20mM NaCl, 0.1mM PMSF, 1mg / mL digitonin, 0.1mg / mL BSA, 1mM CaCl2, 1×Sigma P8340 Protease inhibitor cocktail.

[0017] Preferably, in step S6, the sequence of the initial adapter is as shown in SEQ ID NO: 1, and the sequence of the UMI adapter is as shown in SEQ ID NO: 2, which are specifically as follows:

[0018] Initial linker: 5'-ACACTCTTTCCCTACACGACGCTCTTCCGATC-3' (SEQ ID NO: 1);

[0019] UMI linker: 5'-AGTCANNNNNNGATCGGAAGAGCACACGTCTGAACTCCAGTC-3' (SEQ ID NO: 2).

[0020] Preferably, in step S6, the UMI adapter has 12 random bases at both ends, with a complexity of 4^12=16,777,216, which can distinguish 0.5×4^12=8,388,608 interphase cells, or 4^11=4,194,304 metaphase cells.

[0021] Compared with the prior art, the beneficial effects of the present invention are as follows: the present invention purifies metaphase chromosomes through centrifugal filter membrane integration, and utilizes the developed unique MNase buffer to effectively digest metaphase chromosomes with MNase at low temperature (4°C) and protect nucleosomes in situ, thereby stably obtaining single nucleosomes on metaphase cell chromosomes, and combining quantitative library construction and high-throughput sequencing technology to achieve genome-wide localization and quantification of nucleosomes on metaphase chromosomes, with robust results and accurate quantification, and can be widely used in quantitative studies of nucleosomes on various metaphase chromosomes, providing a new methodological basis for research in related fields, such as histone variants and modifications. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 Flow chart of the preparation of nucleosomes on metaphase chromosomes in the examples.

[0023] Figure 2Optimization of MNase buffer and MNase digestion test results in the examples; (A) Low concentration (0.1 mM) of calcium ions can effectively activate the activity of MNase, which can be used for chromosome digestion. Calcium ions are a necessary condition for maintaining MNase activity. MNase cannot digest chromosomes without calcium ions (lane 2); the calcium ion concentration contained in the conventional MNase buffer is as high as 5 mM, which causes chromosome condensation and makes digestion difficult. It is necessary to reduce the calcium ion concentration and ensure sufficient MNase activity; in order to reduce the calcium ion concentration and ensure the digestion effect, the digestion conditions with lower calcium ion concentrations are explored; the experimental results show that MNase has sufficient activity for chromosome digestion under conditions as low as 0.1 mM calcium ions (lane 3); (B) Low temperature of 4°C can stably maintain the morphological structure of chromosomes. The conventional 37°C digestion reaction conditions, the higher temperature will cause the chromosome structure to expand and disperse (right figure), causing the nucleosome structure to be distorted. Spermine and spermidine components can effectively maintain the structure of chromosomes. The use of the above components and 0.1 mM Ca 2+ MNase buffer and long-term reaction at 4°C, the chromosome and nucleosome structure are not destroyed (left); (C) Metaphase chromosomes at 4°C, 0.1 Mm Ca 2+ MNase buffer with 3000 gel units / mL can effectively cleave metaphase chromosomes into single nucleosomes (lane 2) after 12 hours.

[0024] Figure 3 This is a linker containing a random hexamer UMI in the embodiment. The upper red part in the left picture is chain A, and the lower part is chain B. The purple area is the random hexamer UMI, and the orange part is the UMI marker.

[0025] Figure 4 Length distribution diagram of single nucleosome data reads of metaphase chromosomes in the embodiment.

[0026] Figure 5 This is a schematic diagram for visualizing the sequencing results in the examples. DETAILED DESCRIPTION

[0027] In order to make the purpose, technical solution and advantages of the present invention clearer, the technical solution of the present invention will be clearly and completely described below in combination with the embodiments and drawings. Obviously, the described embodiments are part of the embodiments of the present invention, not all of them, and all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0028] Example 1: Preparation of metaphase chromosome mononucleosomes from NIH-3T3 cells

[0029] The method flow for efficiently obtaining nucleosomes on metaphase chromosomes is as follows Figure 1 As shown, the mononucleosomes are prepared by taking the in vitro cultured cell line NIH-3T3 cells as an example, and the specific steps are as follows:

[0030] S1. Preparation of metaphase cells: NIH-3T3 cells were cultured in vitro. After the cell growth density reached 80%, 100 ng / mL colchicine was added and cultured for 12 hours to obtain synchronized metaphase cells.

[0031] S2. Collect metaphase cells: discard the culture medium, add 5 mL 1× PBS, tap the culture dish gently to make the metaphase cells fall off, and collect them into a 15 mL centrifuge tube.

[0032] S3. Hypotonicity: Centrifuge (RT, 200g, 2 min), discard the supernatant; resuspend the cells in 10 mL 75 mM potassium chloride solution and incubate at room temperature for 30 min.

[0033] S4. Collect metaphase chromosomes: After hypotonicity ends, centrifuge (4°C, 600g, 5min), resuspend the precipitate with 1×PAbuffer, homogenize thoroughly with a homogenizer, filter through 10μm and 5μm filter membranes in turn, and centrifuge to obtain metaphase chromosome precipitate, wherein 1×PAbuffer is prepared from 10×PAbuffer, and the compositions of 10×PA buffer and 1×PAbuffer are shown in Tables 1 and 2.

[0034] Table 1

[0035] <![CDATA[ddH2O]]> - 17.4mL 1M Tris-HCl pH7.5 150mM 6mL 0.5M EDTA pH 8.0 20mM 1.6mL 2.5M KCl 1800mM 12.8mL 5M NaCl 200mM 1.6mL 328.6mM EGTA 5mM 608.6μL

[0036] Table 2

[0037] <![CDATA[ddH2O]]> 13.4mL 10×PA buffer 1.5mL PMSF 30μL Spermidine(0.5mM) 75μL Spermine(0.2mM) 12.4μL Digitonin 0.015g

[0038] S5. The metaphase chromosomes purified above were resuspended in optimized MNase buffer. The composition of MNase buffer is shown in Table 3. MNase with a final concentration of 3,000 gel units / mL was added. The enzyme digestion reaction was carried out at 4°C for 12 h. The mononucleosome preparation with normal chromosome structure was achieved by optimizing MNase buffer and MNase digestion conditions, such as Figure 2 shown. Figure 2In A, calcium ions are a prerequisite for MNase to maintain its activity. Extra calcium ions alone make the structure of chromosomes more condensed, making enzyme cutting difficult. In order to limit the calcium ion concentration and ensure the enzyme cutting effect, the optimal calcium ion concentration experiment shows that MNase can be sufficiently active for chromosome enzyme cutting under conditions as low as 0.1mM calcium ions; Figure 2 In B, the conventional restriction digestion reaction conditions of 37°C and 30 min will destroy the chromosome structure and distort the nucleosome structure. Spermine and spermidine components can effectively maintain the chromosome structure. 2+ MNase buffer and react for a long time at 4°C, the chromosome and nucleosome structures are not destroyed; Figure 2 C, metaphase chromosomes can be effectively cleaved into mononucleosomes at 4°C and 3,000 gel units / mL for 12 hours.

[0039] Table 3

[0040]

[0041]

[0042] S6, MNase digestion effect reached stability in 12h, at which time the metaphase chromosomes were prepared by completing appropriate enzyme reactions in MNase buffer to obtain mononucleosomes ( Figure 2 ).

[0043] After S7 and MNase digestion, 20 mM EDTA was used to terminate the reaction, and the phenol-chloroform extraction method was used to extract DNA, which contained the mononucleosome positioning information and was used for further gel electrophoresis and whole genome sequencing analysis.

[0044] Example 2: Quantitative analysis of mononucleosome localization

[0045] For the mononucleosomes prepared in Example 1, about 1 ng of DNA can be taken from each sample to prepare a sequencing library. The specific steps are as follows:

[0046] S1. For quantitative DNA library construction, an adapter containing a random hexamer (N6) index is designed as a UMI for accurate quantification. The combined double-ends of the adapter are 12 random bases with a complexity of 4^12=16,777,216, which can distinguish 0.5×4^12=8,388,608 interphase cells or 4^11=4,194,304 metaphase cells. The adapter used for library construction is as follows: Figure 3Shown: initial linker (chain A): 5′-ACACTCTTTCCCTACACGACGCTCTTCCGATC-3;

[0047] UMI adapter (chain B): 5'-AGTCANNNNNNGATCGGAAGAGCACACGTCTGAACTCCAGTC-3'; the upper left red part is chain A, the lower part is chain B, the purple area is the random hexamer UMI, and the orange part is the UMI marker. After fill-in and T-tailing, the 3 ends of chain A are filled and dTTP is added to prepare a complete Y-shaped adapter for subsequent quantitative library construction.

[0048] S2, UMI connector preparation process is as follows:

[0049] S2.1. Chain A and chain B complete complementary pairing by low temperature annealing. The reaction system and conditions are shown in Tables 4 and 5:

[0050] 10×anneal buffer: 100mM Tris-HCI, pH 7.5, 1M NaCl, 10mM EDTA, pH 8.0.

[0051] Table 4

[0052]

[0053]

[0054] Table 5

[0055] step cycle temperature time first step 1 95℃ 5min Step 2 70 95℃(-1℃ / cycle) 1min Step 3 1 4℃ maintain

[0056] S2.2, perform a leveling reaction, the reaction system and conditions are shown in Tables 6 and 7:

[0057] Table 6

[0058] Components volume Final concentration Annealed product (10μM) 50μL 5μM 10×NEBuffer 10μL 1× Klenow (5 units / μL) 2μL 0.1units / μL dNTP(10mM) 5μL 500μM NFW Make up to 100 μL -

[0059] Table 7

[0060] step cycle temperature time first step 1 25℃ 15min

[0061] S2.3, add EDTA to a final concentration of 10mM, and treat at 75°C for 20min to stop the reaction. S2.4, perform tailing reaction, the reaction system and conditions are shown in Tables 8 and 9:

[0062] Table 8

[0063] Components volume Final concentration 2×dT-tailing Buffer 25μL 1× 25μL DNA fragment (5μM) 10μL 5μM dTTP (10 mM) 1μL 0.2mM Klenow (5 units / μL) 1μL 0.1units / μL NFW Make up to 50 μL -

[0064] Table 9

[0065] step cycle temperature time first step 1 72℃ 2h

[0066] S2.5. After treatment with Proteinase K, extract DNA using the phenol-chloroform extraction method and dissolve the linker with NFW water.

[0067] S3, using the above UMI adapter and Ultra TM IIDNA library kit library preparation kit for library preparation. After DNA2200 electrophoresis analysis quality test and confirmation, sequencing was completed on a commonly used second-generation sequencing platform. Reads length distribution analysis was performed based on the data obtained from high-throughput sequencing. The peak of reads length was around 147bp, which is a typical single nucleosome length, proving the reliability and stability of this method ( Figure 4 ).

[0068] S4. The UMI information added during the library construction process can help reduce the high false signals in the original data in bioinformatics analysis, and increase the number of valid reads after deduplication, significantly improving the accuracy of the data ( Figure 5 ), the UMI-based method accurately quantifies the nucleosome distribution. In the example of two reads distribution, the original data information can obtain the position information of the nucleosome, but it causes an error of high signal. Direct deduplication without UMI will lead to the loss of nucleosome information.

[0069] The above is only a preferred embodiment of the present invention, but the present invention should not be limited to the contents disclosed in this embodiment. Any equivalent or modification completed without departing from the spirit disclosed in the present invention shall fall within the scope of protection of the present invention.

Claims

1. A method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes, characterized in that: The following steps are involved: S1. Prepare and enrich metaphase cells, and purify metaphase chromosomes by centrifugal filter integration; S2. Use the independently developed and optimized MNase buffer to resuspend the metaphase chromosome pellet, protect the metaphase chromosome structure, and keep the nucleosome position unchanged; S3, add MNase enzyme with a final concentration of 1000-3,000 gel units / mL and a chromosome concentration of 100-1000 ng, and perform the enzyme digestion reaction at 4°C for 12-24 hours to achieve uniform enzyme digestion of metaphase chromosomes; S4, adding EDTA at a final concentration of 20 mM to terminate the enzyme digestion reaction and obtain a mononucleosome suspension of metaphase chromosomes; S5. Use a DNA extraction kit to extract and quantify DNA; S6. The initial adapter is connected to the index containing random hexamer to obtain the UMI adapter, and the library is constructed by combining with the DNA library construction kit. The obtained library is sequenced and analyzed after passing the quality inspection.

2. The method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes according to claim 1, characterized in that: In step S1, the membrane of the enriched metaphase cells is broken by a homogenizer, and the obtained homogenate is filtered through 10 μm and 5 μm filter membranes, centrifuged, and the precipitate is collected to obtain pure metaphase chromosomes.

3. The method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes according to claim 2, characterized in that: In step S1, the centrifugation conditions are 4°C, 1750g, and 10 min.

4. The method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes according to claim 1, characterized in that: In step S2, the formula of the MNase buffer is: 50mM Tris-HCl, 0.2mM spermine, 0.5mM spermidine, 80mM KCl, 20mM NaCl, 0.1mM PMSF, 1mg / mL digitonin, 0.1mg / mL BSA, 1mM CaCl2, and 1×Sigma P8340 Protease inhibitor cocktail.

5. The method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes according to claim 1, characterized in that: In step S6, the sequence of the initial adapter is shown as SEQ ID NO: 1, and the sequence of the UMI adapter is shown as SEQ ID NO:

2.

6. The method for efficiently obtaining the whole genome positioning and quantification of nucleosomes on metaphase chromosomes according to claim 5, characterized in that: In step S6, the UMI adapter has 12 random bases at both ends, with a complexity of 4^12=16,777,216, which can distinguish 0.5×4^12=8,388,608 interphase cells, or 4^11=4,194,304 metaphase cells.

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