CN107841533B - Chromatin fragmentation method and application thereof - Google Patents
Chromatin fragmentation method and application thereof Download PDFInfo
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- CN107841533B CN107841533B CN201711113635.2A CN201711113635A CN107841533B CN 107841533 B CN107841533 B CN 107841533B CN 201711113635 A CN201711113635 A CN 201711113635A CN 107841533 B CN107841533 B CN 107841533B
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Abstract
The invention provides a chromatin fragmentation method and application thereof, wherein the method comprises the following steps: formaldehyde crosslinking, buffer solution treatment, micrococcus nuclease digestion and ultrasonic disruption. The invention combines two means of ultrasonic disruption and micrococcus enzyme digestion, develops the buffer solution suitable for the method, can avoid the problems of chromatin fragment dispersion and enzyme digestion sequence specificity, reduces the probability of protein denaturation, and saves time and reagents. The method can quickly and stably obtain uniform chromatin fragments and is used for library construction of ChIP-Seq or chromatin co-immunoprecipitation, and has wide application prospect and great market value.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a chromatin disruption method and application thereof
Background
The chromatin-immunoprecipitation (ChIP) technology can truly and completely reflect the regulation and control information of target protein bound on a DNA sequence, and its basic principle: fixing the activities of all DNA binding proteins in cells by formaldehyde crosslinking and other modes at specific time points, randomly cutting chromatin by subsequent cell lysis and chromosome separation, utilizing the specific recognition reaction of antigen and antibody, precipitating DNA fragments combined with target proteins, releasing the DNA fragments of the binding proteins by decrosslinking, and performing downstream analysis after purification. ChIP-Seq is a deep sequencing technology which combines a high-throughput sequencing technology and a ChIP experiment to analyze DNA binding protein sites, DNA methylation, histone modification and the like in the whole genome range. The method is generally used for researching transcription factor binding sites or histone specificity modification sites, is helpful for understanding the mutual regulation and control among genes and the functional structure of chromosomes, and has the advantages of high resolution (base level), high sensitivity (less sample amount is needed), strong flexibility and the like. At present, with the advantages of continuous reduction of sequencing cost, rapid increase of throughput and the like, ChIP-Seq basically replaces ChIP-ChIP to become a main technology for researching the in vivo binding target of DNA binding proteins such as transcription factors, RNA polymerase, nucleosomes and the like.
In the ChIP-Seq technology, the construction of the library is a very important step, and the quality of the library construction directly influences the accuracy of the sequencing result. It is important to be able to rapidly and stably break chromatin into fragments of uniform size during library construction. CN106834208A discloses an ultrasonic disruption method of naked mole tissue in chromatin co-immunoprecipitation, which comprises the steps of adding a collagenase II solution, a hyaluronidase solution and a mixed solution of DPBS buffer solution in a volume ratio of 1:1:1 into the naked mole tissue, oscillating and digesting the tissue at 37 ℃, centrifuging to remove supernatant, and then carrying out conventional ultrasonic disruption. CN104651506A discloses a method for histone modification chromosome co-immunoprecipitation applied to a tissue sample, which comprises the steps of firstly, preprocessing the tissue sample; then grinding in ice bath, filtering with sterile gauze, collecting cells in filtrate, and suspending in water bath in digestion buffer solution; adding micrococcal nuclease into the cells added with the digestion buffer solution for enzyme digestion, and adding an enzyme digestion stop solution with the same amount as the micrococcal nuclease after enzyme digestion; performing high-frequency ultrasonic treatment and centrifugal treatment in ice bath to obtain a broken chromatin solution; adding a PBS buffer solution containing bovine serum albumin into the ProteinA/G, adding an antibody for incubation, adding a broken chromatin solution, and incubating overnight; washing the magnetic beads with a high salt solution for a plurality of times on a magnetic frame, eluting DNA on the magnetic beads with a TE solution after washing, purifying and extracting the DNA with a purification solution, and dissolving the DNA with ultrapure water.
One method commonly used for chromatin fragmentation today is to obtain chromatin fragments of a certain size by mechanical disruption, i.e. sonication. The ultrasound parameters used vary according to the cell sample and the size of the fragments required for the banking. The disadvantages mainly include the following: when a smaller fragment needs to be obtained (generally, the fragment is smaller than 200 bp), the ultrasonic time and the ultrasonic power need to be increased, so that the probability of protein denaturation is greatly increased, and the subsequent specific binding of an antibody and a protein is influenced. ② the size of DNA fragment obtained by ultrasonic wave is relatively concentrated on a certain position, but the size dispersion range is too wide, which can lead to one of the following: in order to meet the requirement of building a library, the amount of the DNA needs to be greatly increased; the second step is as follows: because the DNA obtained by ultrasonic is wide in dispersion, and the size range of the DNA selected during library construction is relatively narrow, the sample waste can be caused, and the preference of the DNA selected during library construction can be caused, so that the final sequencing result is influenced.
Another common method for chromatin fragmentation is to obtain chromatin fragments of a certain size by using an enzymatic digestion method, the commonly used enzyme is micrococcus Nuclease (MNase), which is a Nuclease that only degrades nucleosome junction DNA, and can fragment chromatin since DNA at nucleosomes is protected by histones and not degraded. The main disadvantages of using micrococcal nuclease alone as a disruption means are the following: micrococcal nucleases have a degree of sequence specificity in cleaving DNA, and thus may present sequence preference problems in disrupting chromatin. Secondly, the enzyme amount is difficult to control, the phenomenon of nonuniform enzyme digestion is easily caused, namely chromatin fragments with different sizes are generated, the size is about 150bp when a single nucleosome is cut off, and the size is about 300bp when two nucleosomes are cut off, so when the enzyme is simply used as a chromatin crushing means, the phenomenon of mixing the fragments with different sizes is easily caused.
Although the methods used at present can barely meet the basic requirements of library construction, the methods all have the following: large sample amount, high cost, long time consumption, sequence preferential fragmentation and the like, so a chromatin fragmentation method which can be quickly and conveniently suitable for laboratory researchers is needed.
Disclosure of Invention
Aiming at the defects and practical requirements of the prior art, the invention provides a chromatin fragmentation method and application thereof, wherein the method is simple and efficient, ultrasonic fragmentation and micrococcus nuclease digestion are combined, a corresponding buffer solution is searched out, the problems of protein denaturation, enzyme digestion sequence bias and the like are avoided, a uniform and stable product is obtained, and the requirements of ChIP-Seq library construction are met.
In order to achieve the purpose, the invention adopts the following technical scheme:
in one aspect, the invention provides a method of chromatin disruption comprising the steps of:
(1) fixing the cells by a formaldehyde crosslinking method;
(2) centrifuging the cells and treating the cells with a buffer solution;
(3) the treated solution was digested with micrococcal nuclease and sonicated.
Through a large amount of experimental researches and long-time groping, the inventor discovers that chromatin is crushed by mixing and cutting through ultrasound and enzyme, overcomes the defects of the prior art, can avoid the problems of chromatin fragment dispersion and enzyme cutting sequence specificity, reduces the probability of protein denaturation, saves time and reagents, reduces the time of ultrasound, reduces the power of ultrasound, better crushes chromatin, and meets the requirements of ChIP-Seq library establishment.
Preferably, the buffer in step (2) includes any one or a combination of at least two of buffer a, buffer CF1, buffer CF2, buffer N1 and buffer N2, for example, a combination of buffer a and buffer CF1, a combination of buffer CF1 and buffer CF2, a combination of buffer N1 and buffer N2 or a combination of buffer a, buffer CF1, buffer CF2, buffer N1 and buffer N2, preferably a combination of buffer a, buffer CF1, buffer CF2, buffer N1 and buffer N2.
Preferably, the buffer a comprises: hepes (4-hydroxyethylpiperazine ethanesulfonic acid), MgCl2Any one or a combination of at least two of DTT (dithiothreitol) and PMSF (phenylmethylsulfonyl fluoride), and examples thereof include Hepes and MgCl2Combination of (A), DTT and MgCl2A combination of DTT and PMSF or Hepes, MgCl2DTT and PMSF, preferably Hepes, MgCl2A combination of DTT and PMSF.
Preferably, the Hepes has a pH of 7.8-8, which may be, for example, 7.8, 7.9 or 8.0, preferably 7.9.
Preferably, the final concentration of Hepes is 8-12mM, for example, may be 8mM, 9mM, 10mM, 11mM or 12mM, preferably 10 mM.
Preferably, the MgCl2The final concentration of (B) is 0.5 to 2mM, and may be, for example, 0.5mM, 0.8mM, 1mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM or 2mM, preferably 1.5 mM.
Preferably, the final concentration of DTT is 0.5-2mM, and may be, for example, 0.5mM, 0.8mM, 1mM, 1.2mM, 1.4mM, 1.6mM, 1.8mM, or 2mM, preferably 1 mM.
Preferably, the final concentration of PMSF is 0.2-1mM, and may be, for example, 0.2mM, 0.4mM, 0.5mM, 0.6mM, 0.8mM, or 1mM, preferably 0.5 mM.
In the invention, the inventor selects the buffer solution with specific components and concentration through long-term experimental investigation and verification, can adapt to and match the method for breaking chromatin by mixing ultrasonic and enzyme digestion, helps to exert the optimal action effect of the method, avoids the problems of chromatin fragment dispersion and enzyme digestion sequence specificity, reduces the probability of protein denaturation, and saves time and reagents.
Preferably, the buffer CF1 includes any one or a combination of at least two of KCl, NP40(Nonidet P40, ethylphenylpolyethylene glycol) and buffer a, and may be, for example, a combination of buffer a and KCl, a combination of buffer a and NP40 or a combination of buffer A, NP40 and KCl, preferably a combination of buffer A, NP40 and KCl.
Preferably, the final concentration of KCl is 5-15mM, and may be, for example, 5mM, 6mM, 7mM, 8mM, 9mM, 10mM, 11mM, 12mM, 13mM or 15mM, preferably 10 mM.
Preferably, the mass percent of NP40 is 0.1-5%, and may be, for example, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.8%, 1%, 1.5%, 2%, 2.5%, 3.5%, 4%, or 5%, preferably 1%.
Preferably, the buffer CF2 comprises any one or a combination of at least two of KCl, NaCl, glycerol, NP40 and buffer a, and may be, for example, a combination of KCl and NaCl, a combination of NaCl and glycerol, a combination of glycerol and NP0 or a combination of KCl, NaCl, glycerol, NP40 and buffer a, preferably a combination of KCl, NaCl, glycerol, NP40 and buffer a.
Preferably, the final concentration of KCl is 5-15mM, and may be, for example, 5mM, 8mM, 10mM, 12mM, 14mM or 15mM, preferably 10 mM.
Preferably, the mass percent of the NP40 is 0.1-1%, and may be, for example, 0.1%, 0.2%, 0.3%, 0.5%, 0.8%, or 1%, preferably 0.5%.
Preferably, the final concentration of NaCl is 50-100mM, and may for example be 50mM, 60mM, 70mM, 75mM, 80mM, 85mM, 90mM, 95mM or 100mM, preferably 75 mM.
Preferably, the glycerol is present in a mass percentage of 5 to 15%, for example 5%, 7%, 8%, 10%, 11%, 12% or 15%, preferably 10%.
Preferably, the buffer N1 comprises: tris, NaCl, sucrose, MgCl2KCl and CaCl2Any one or a combination of at least two of them, for example, a combination of Tris and NaCl, sucrose, MgCl2And KCl or Tris, NaCl, sucrose, MgCl2KCl and CaCl2Preferably Tris, NaCl, sucrose, MgCl2KCl and CaCl2Combinations of (a) and (b).
Preferably, the pH of the buffer N1 is 6.0-9.0, and may be, for example, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5 or 9.0, preferably 8.0.
Preferably, the final concentration of Tris is 13-20mM, e.g.may be, preferably is 15 mM.
Preferably, the final concentration of NaCl is 10-20mM, and may be, for example, 10mM, 13mM, 15mM, 18mM or 20mM, preferably 15 mM.
Preferably, the sucrose is present in an amount of 1-5% by mass, for example 1%, 2%, 3%, 4% or 5%, preferably 3%.
Preferably, the MgCl2The final concentration of (B) is 3 to 10mM, and may be, for example, 3mM, 4mM, 5mM, 6mM, 7mM, 8mM or 10mM, preferably 5 mM.
Preferably, the final concentration of KCl is 4-6mM, and may be, for example, 4mM, 5mM or 6mM, preferably 5 mM.
Preferably, the CaCl2The final concentration of (B) is 1 to 2mM, and may be, for example, 1mM, 1.2mM, 1.5mM, 1.8mM or 2mM, preferably 1.5 mM.
Preferably, the buffer N2 includes NP40 and buffer N1.
Preferably, the mass percent of NP40 is 0.5-3%, and may be, for example, 0.5%, 1%, 1.5%, 2%, 2.5%, or 3%, preferably 1%.
Preferably, the DNA concentration of the solution after the treatment in step (3) is 0.5 to 3. mu.g/. mu.L, and may be, for example, 0.5. mu.g/. mu.L, 0.8. mu.g/. mu.L, 1. mu.g/. mu.L, 1.5. mu.g/. mu.L, 1.8. mu.g/. mu.L, 2. mu.g/. mu.L, 2.5. mu.g/. mu.L, or 3. mu.g/. mu.L, preferably 1. mu.g/. mu.L.
Preferably, the micrococcal nuclease is added in the step (3) in an amount of micrococcal nuclease: the DNA (U: μ g) is 1 (10-30), and may be, for example, 1:10, 1:15, 1:20, 1:25 or 1:30, preferably 1: 20.
Preferably, the temperature of the digestion in step (3) is 30-50 ℃, for example 30 ℃, 37 ℃, 40 ℃, 45 ℃ or 50 ℃, preferably 37 ℃.
Preferably, the digestion time in step (3) is 10-30min, for example 10min, 15min, 20min or 30min, preferably 15 min.
Preferably, the step of the ultrasound in the step (2) is intermittently started for 5s and paused for 30 s.
Preferably, the frequency of the ultrasound is 20 kHz.
Preferably, the number of cycles of ultrasound is 3-5, for example, it may be 3, 4 or 5, preferably 4.
As a preferred embodiment, a method for chromatin fragmentation, comprising the steps of:
(1) fixing the cells by a formaldehyde crosslinking method;
(2) centrifuging the cells, and treating the cells with buffer A, CF1, CF2, N1 and N2 in sequence;
the buffer solution A comprises: hepes at a final concentration of 8-12mM, MgCl at a final concentration of 0.5-2mM2DTT with the final concentration of 0.5-2mM and PMSF with the final concentration of 0.2-1mM, and the pH value is 7.8-8;
the buffer solution CF1 comprises KCl with the final concentration of 5-15mM, NP40 with the mass percent of 0.1-5% and buffer solution A;
the buffer solution CF2 comprises KCl with the final concentration of 5-15mM, NaCl with the final concentration of 50-100mM, glycerol with the mass percent of 5-15%, NP40 with the mass percent of 0.1-1% and buffer solution A;
buffer N1 included: 13-20mM Tris, 10-20mM NaCl, 1-5% sucrose, 3-10mM MgCl24-6mM KCl and 1-2mM CaCl2；
The buffer N2 comprises buffer N1 and NP40 with the mass percent of 0.5-3%.
(3) Digesting the treated solution by using micrococcal nuclease and carrying out ultrasonic crushing, wherein the digestion temperature is 30-50 ℃, the digestion time is 10-30min, the DNA content of the treated solution is 0.5-3 mug/mu L, and the addition amount of the micrococcal nuclease is as follows: the DNA (U: mug) is (10-30):1, the ultrasound step is intermittently started for 5s, temporarily stopped for 30s, the number of cycles is 3-5, and the ultrasound frequency is 20 kHz.
In a second aspect, the invention provides a kit comprising buffer a, buffer CF1, buffer CF2, buffer N1, buffer N2 and a micrococcal nuclease.
Preferably, the kit is for chromatin disruption.
In a third aspect, the present invention provides a method according to the first aspect and/or a kit according to the second aspect for library construction and/or chromatin co-immunoprecipitation of ChIP-Seq.
Compared with the prior art, the invention has the following beneficial effects:
(1) according to the chromatin fragmentation method provided by the invention, through combining ultrasonic fragmentation with micrococcal nuclease digestion, and developing a buffer solution suitable for the method, the problems of chromatin fragment dispersion and enzyme digestion sequence specificity can be avoided, the probability of protein denaturation is reduced, time and reagents are saved, compared with the method of singly using microspherical nuclease for fragmentation, the using amount of enzyme is reduced by 30% -50%, the power used by ultrasonic and the total time used by ultrasonic are reduced by 20% -40%, and the required initial sample is reduced by 25% -45%;
(2) the buffer solution formula used by the chromatin fragmentation method provided by the invention is a common medicine in biochemical laboratories, is convenient to use, can quickly and stably obtain uniform chromatin fragments, is applied to library construction of ChIP-Seq or a chromatin co-immunoprecipitation experiment, and has wide application prospect and market value.
Drawings
FIG. 1 is an agarose gel electrophoresis image of example 1 of the present invention;
FIG. 2 is an agarose gel electrophoresis image of comparative example 1 of the present invention;
FIG. 3 is an agarose gel electrophoresis image of comparative example 2 of the present invention;
FIG. 4 is an agarose gel electrophoresis image of comparative example 3 of the present invention.
Detailed Description
To further illustrate the technical means and effects of the present invention, the following further describes the technical solutions of the present invention by way of specific embodiments with reference to the drawings, but the present invention is not limited to the scope of the embodiments.
Example 1
1. Inoculating cells on a 15cm cell plate, sucking the culture medium to the remaining 15mL when the cell density is 60-80%, adding 1mL of 16% formaldehyde to make the final concentration of formaldehyde be 1%, fixing at room temperature for 10min, adding 950 μ L of 2.5M glycine to stop the formaldehyde action, and keeping at room temperature for 10 min;
2. pouring out the culture medium, washing twice with cold 10mL PBS, scraping the cells, putting into a 1.5mL EP tube, centrifuging at 4 ℃ for 2min at 250g, sucking off the supernatant, recording the cell volume, adding a buffer solution A with 5 times of the cell volume for heavy suspension, centrifuging at 4 ℃ for 2min at 250g, sucking off the supernatant, adding a buffer solution CF1 with 2 times of the cell volume for heavy suspension, centrifuging at 500g for 2min, sucking off the supernatant, adding a buffer solution CF2 with 1 times of the cell volume for heavy suspension, standing on ice for 10min, centrifuging at 500g for 2min, sucking off the supernatant, adding 1mL of a buffer solution N1 for heavy suspension, centrifuging at 500g for 2min, sucking off the supernatant, and adding 600 mu L of a buffer solution N2 for heavy suspension;
the buffer solution A comprises: hepes at a final concentration of 10mM, MgCl at a final concentration of 1.5mM2DTT with a final concentration of 1mM and PMSF with a final concentration of 0.5mM, pH 7.9;
the buffer solution CF1 comprises KCl with the final concentration of 10mM, NP40 with the mass percent of 1% and a buffer solution A;
the buffer solution CF2 comprises KCl with the final concentration of 10mM, NaCl with the final concentration of 75mM, glycerol with the mass percent of 10%, NP40 with the mass percent of 0.5% and a buffer solution A;
buffer N1 included: 15mM Tris, 15mM NaCl, 3% sucrose, 5mM MgCl25mM KCl and 1.5mM CaCl2；
The buffer N2 comprises buffer N1 and NP40 with the mass percent of 1%.
3. Adding 390 mu L of 2M NaCl into 10 mu L of the heavy suspension of the buffer solution N2, fully mixing uniformly, taking 2 mu L of the mixture to measure the DNA concentration of the mixture, and calculating the DNA concentration in the buffer solution N2 for a micrococcus nuclease digested sample, wherein the DNA concentration is ensured to be 1 mu g/mu L; taking 300 mu L of the resuspension of the buffer solution N2 to a new EP tube, and adding micrococcal nuclease, wherein the addition amount of the micrococcal nuclease is micrococcal nuclease: DNA (U: mug) is 1:20, ultrasonic treatment is carried out after digestion is carried out for 15min at 37 ℃, the ultrasonic frequency is 20kHz, the parameters are 15s/on and 30s/off, and 4 cycles of ultrasonic treatment are carried out; the sample after the ultrasound treatment can be subjected to co-immunoprecipitation and DNA fragment recovery, and these steps are common to the conventional techniques, and therefore are not described herein.
The DNA fragment thus obtained was subjected to 1.5% agarose gel electrophoresis, and the results are shown in FIG. 1.
Example 2
1. Inoculating cells on a 15cm cell plate, sucking the culture medium to the remaining 15mL when the cell density is 60-80%, adding 1mL of 16% formaldehyde to make the final concentration of formaldehyde be 1%, fixing at room temperature for 10min, adding 950 μ L of 2.5M glycine to stop the formaldehyde action, and keeping at room temperature for 10 min;
2. pouring out the culture medium, washing twice with cold 10mL PBS, scraping the cells, putting the cells into a 1.5mLEP tube, centrifuging at 4 ℃ for 2min at 250g, sucking off the supernatant, recording the cell volume, adding a buffer solution A with 5 times of the cell volume for heavy suspension, centrifuging at 4 ℃ for 2min at 250g, sucking off the supernatant, adding a buffer solution CF1 with 2 times of the cell volume for heavy suspension, centrifuging at 500g for 2min, sucking off the supernatant, adding a buffer solution CF2 with 1 time of the cell volume for heavy suspension, standing on ice for 10min, centrifuging at 500g for 2min, sucking off the supernatant, adding 1mL buffer solution N1 for heavy suspension, centrifuging at 500g for 2min, sucking off the supernatant, and adding 600 mu L buffer solution N2 for heavy suspension;
the buffer solution A comprises: hepes at a final concentration of 8mM, MgCl at a final concentration of 0.5mM2DTT with a final concentration of 0.5-2mM and PMSF with a final concentration of 0.2-1mM, pH 7.8;
the buffer solution CF1 comprises KCl with the final concentration of 5mM, NP40 with the mass percent of 0.1% and a buffer solution A;
the buffer solution CF2 comprises KCl with the final concentration of 5mM, NaCl with the final concentration of 50mM, glycerol with the mass percent of 5%, NP40 with the mass percent of 0.1% and a buffer solution A;
buffer N1 included: 15mM Tris, 15mM NaCl, 3% sucrose, 5mM MgCl25mM KCl and 1.5mM CaCl2；
The buffer N2 comprises buffer N1 and NP40 with the mass percent of 0.5%.
3. Adding 390 mu L of 2M NaCl into 10 mu L of the heavy suspension of the buffer solution N2, fully mixing uniformly, taking 2 mu L of the mixture to measure the DNA concentration of the mixture, and calculating the DNA concentration in the buffer solution N2 for a micrococcus nuclease digested sample, wherein the DNA concentration is ensured to be 0.5 mu g/mu L; taking 300 mu L of the resuspension of the buffer solution N2 to a new EP tube, and adding micrococcal nuclease, wherein the addition amount of the micrococcal nuclease is micrococcal nuclease: DNA (U: mug) is 1:10, ultrasonic treatment is carried out after digestion is carried out for 10min at 30 ℃, the frequency is 20kHz, the parameters are 15s/on and 30s/off, and 3 cycles of ultrasonic treatment are carried out; the sample after the ultrasound treatment can be subjected to co-immunoprecipitation and DNA fragment recovery, and these steps are common to the conventional techniques, and therefore are not described herein.
Example 3
1. Cells were seeded on 15cm cell plates, and when the cell density reached 60% -80%, the medium was aspirated to the remaining 15mL, 1mL of 16% formaldehyde was added to give a final formaldehyde concentration of 1%, and after fixing at room temperature for 10 minutes, 950 μ L of 2.5M glycine was added to terminate the formaldehyde action, at room temperature for 10 minutes.
2. Pouring out the culture medium, washing twice with cold 10mL PBS, scraping the cells, putting the cells into a 1.5mLEP tube, centrifuging at 4 ℃ for 2min at 250g, sucking off the supernatant, recording the cell volume, adding a buffer solution A with 5 times of the cell volume for heavy suspension, centrifuging at 4 ℃ for 2min at 250g, sucking off the supernatant, adding a buffer solution CF1 with 2 times of the cell volume for heavy suspension, centrifuging at 500g for 2min, sucking off the supernatant, adding a buffer solution CF2 with 1 time of the cell volume for heavy suspension, standing on ice for 10min, centrifuging at 500g for 2min, sucking off the supernatant, adding 1mL buffer solution N1 for heavy suspension, centrifuging at 500g for 2min, sucking off the supernatant, and adding 600 mu L buffer solution N2 for heavy suspension;
the buffer solution A comprises: hepes at a final concentration of 12mM, MgCl at a final concentration of 2mM2DTT with a final concentration of 2mM and PMSF with a final concentration of 1mM,the pH is 8;
the buffer solution CF1 comprises KCl with the final concentration of 15mM, NP40 with the mass percent of 5% and a buffer solution A;
the buffer solution CF2 comprises KCl with the final concentration of 15mM, NaCl with the final concentration of 100mM, glycerol with the mass percent of 15%, NP40 with the mass percent of 1% and a buffer solution A;
buffer N1 included: 20mM Tris, 20mM NaCl, 5% sucrose, 10mM MgCl2KCl of 6mM and CaCl of 2mM2；
The buffer N2 comprises buffer N1 and NP40 with the mass percent of 3%.
3. Adding 390 mu L of 2M NaCl into 10 mu L of the heavy suspension of the buffer solution N2, fully mixing uniformly, taking 2 mu L of the mixture to measure the DNA concentration of the mixture, and calculating the DNA concentration in the buffer solution N2 for a micrococcus nuclease digested sample, wherein the DNA concentration is ensured to be 3 mu g/mu L; taking 300 mu L of the resuspension of the buffer solution N2 to a new EP tube, and adding micrococcal nuclease, wherein the addition amount of the micrococcal nuclease is micrococcal nuclease: DNA (U: mug) is 1:30, ultrasonic treatment is carried out after digestion is carried out for 30min at 50 ℃, the ultrasonic frequency is 20kHz, the parameters are 15s/on and 30s/off, and 5 cycles of ultrasonic treatment are carried out; the sample after the ultrasound treatment can be subjected to co-immunoprecipitation and DNA fragment recovery, and these steps are common to the conventional techniques, and therefore are not described herein.
Comparative example 1
Compared with example 1, except that no Micrococcus nuclease is added for digestion, the ultrasonic parameters are changed to 15s/On and 20s/off, and 6 cycles of ultrasonic treatment are carried out, the other conditions are the same as example 1.
The DNA fragment thus obtained was subjected to 1.5% agarose gel electrophoresis, and the results are shown in FIG. 2.
Comparative example 2
Compared with example 1, except that no ultrasonication was performed, the enzyme digestion conditions were changed to the addition of micrococcal nuclease: the conditions other than DNA (U: μ g)1:5 were the same as in example 1.
The DNA fragment thus obtained was subjected to 1.5% agarose gel electrophoresis, and the results are shown in FIG. 3.
Comparative example 3
Compared with example 1, except that no Micrococcus nuclease is added for digestion, the ultrasonic parameters are changed to 10 cycles, other conditions are the same as example 1.
The DNA fragment thus obtained was subjected to 1.5% agarose gel electrophoresis, and the results are shown in FIG. 4.
Experimental detection
The DNA fragments obtained in examples 1 to 3 and comparative examples 1 to 3 were subjected to 1.5% agarose gel electrophoresis, and the results are shown in FIGS. 1 to 4.
The fragment size required for ChIP-Seq library construction is the size of a single nucleosome fragment, and as can be seen from FIG. 1, when the technical route (ultrasonic + micrococcal nuclease) of the invention is used as a fragment for breaking chromatin, more than 90% of the obtained DNA fragments are single nucleosomes and basically have no diffusion phenomenon, and the gel electrophoresis patterns of example 2 and example 3 are basically consistent with the electrophoresis pattern of example 1.
As can be seen from FIGS. 2 and 4, in comparative examples 1 and 3, after chromatin is broken and de-crosslinked by using the ultrasonic method, even if the number of ultrasonic cycles is increased, the obtained DNA fragments still have serious dispersion after electrophoresis, which affects the quality of subsequent sequencing.
As can be seen from FIG. 3, in comparative example 2, when the Micrococcus nuclease was used as a single means for disrupting chromatin, the amount of the enzyme used was increased, and the size of the obtained DNA fragments was not uniform, which failed to satisfy the requirement of ChIP-Seq library construction.
In conclusion, the chromatin fragmentation method provided by the invention combines ultrasonic fragmentation with microspherical enzyme digestion, and develops the buffer solution suitable for the method, so that the problems of chromatin fragment dispersion and enzyme digestion sequence specificity can be avoided, the probability of protein denaturation is reduced, and time and reagents are saved. The DNA fragments obtained after the chromatin is broken and the chromatin is uncrosslinked by a single ultrasonic method have serious dispersion after electrophoresis, which affects the subsequent sequencing quality. When micrococcal nuclease is used as a single means for disrupting chromatin, the resulting DNA fragments are not uniform in size and cannot meet the requirements for library construction. Compared with the method of purely using microspherical enzyme for crushing, the method has the advantages that the using amount of the enzyme is reduced by 30-50%, the power used by ultrasonic and the total time used by ultrasonic are reduced by 20-40%, the required initial sample is reduced by 25-45%, uniform chromatin fragments can be quickly and stably obtained and applied to library construction of ChIP-Seq or chromatin co-immunoprecipitation experiments, and the method has wide application prospect and market value.
The applicant states that the present invention is illustrated in detail by the above examples, but the present invention is not limited to the above detailed methods, i.e. it is not meant that the present invention must rely on the above detailed methods for its implementation. It should be understood by those skilled in the art that any modification of the present invention, equivalent substitutions of the raw materials of the product of the present invention, addition of auxiliary components, selection of specific modes, etc., are within the scope and disclosure of the present invention.
Claims (5)
1. A method of chromatin disruption comprising the steps of:
(1) fixing the cells by a formaldehyde crosslinking method;
(2) centrifuging the cells and treating the cells with a buffer solution;
(3) digesting the treated solution by using micrococcal nuclease and carrying out ultrasonic disruption;
the buffer solution in the step (2) is a combination of buffer solution A, buffer solution CF1, buffer solution CF2, buffer solution N1 and buffer solution N2;
the buffer solution A is Hepes and MgCl2A combination of DTT and PMSF; the pH value of Hepes is 7.8-8; the final concentration of Hepes is 8-12 mM; said MgCl2To a final concentration of 0.5-2 mM; the final concentration of the DTT is 0.5-2 mM; the final concentration of the PMSF is 0.2-1 mM;
the buffer CF1 is a combination of KCl, NP40 and buffer A; the final concentration of the KCl is 5-15 mM; the mass percent of the NP40 is 0.1-5%;
the buffer solution CF2 is a combination of KCl, NaCl, glycerol, NP40 and buffer solution A; the final concentration of the KCl is 5-15 mM; the mass percent of the NP40 is 0.1-1%; the final concentration of the NaCl is 50-100 mM; the mass percent of the glycerol is 5-15%;
the buffer solution N1 is Tris, NaCl, sucrose or MgCl2 KCl and CaCl2A combination of (1);the pH value of the buffer solution N1 is 6.0-9.0; the final concentration of the Tris is 13-20 mM; the final concentration of the NaCl is 10-20 mM; the mass percentage of the sucrose is 1-5%; said MgCl2The final concentration of (A) is 3-10 mM; the final concentration of the KCl is 4-6 mM; the CaCl is2To a final concentration of 1-2 mM;
the buffer N2 comprises NP40 and buffer N1; the mass percent of the NP40 is 0.5-3%;
the DNA concentration of the treated solution in the step (3) is 0.5-3 mug/muL;
the addition amount of the micrococcal nuclease in the step (3) is that the addition amount of the micrococcal nuclease is 1U, and the addition amount of DNA is 10-30 mu g;
the digestion temperature in the step (3) is 30-50 ℃;
the digestion time in the step (3) is 10-30 min;
the step of the ultrasonic treatment in the step (3) is that the ultrasonic treatment is started intermittently for 5s and is suspended for 30 s;
the frequency of the ultrasound is 20 kHz;
the number of cycles of the ultrasound is 3-5.
2. The method of claim 1,
in the buffer A, the pH of Hepes is 7.9; the final concentration of Hepes is 10 mM; said MgCl2To a final concentration of 1.5 mM; the final concentration of DTT is 1 mM; the final concentration of the PMSF is 0.5 mM;
in the buffer CF1, the final concentration of KCl is 10 mM; the mass percent of the NP40 is 1%;
in the buffer CF2, the final concentration of KCl is 10 mM; the mass percent of the NP40 is 0.5 percent; the final concentration of NaCl is 75 mM; the mass percent of the glycerol is 10 percent;
in the buffer N1, the pH of the buffer N1 is 8.0; the final concentration of Tris is 15 mM; the final concentration of NaCl is 15 mM; the mass percent of the sucrose is 3%; said MgCl2To a final concentration of 5 mM; the final concentration of KCl is 5 mM; the CaCl is2To a final concentration of 1.5 mM;
in the buffer solution N2, the mass percent of the NP40 is 1%.
3. The method according to claim 2, wherein the DNA concentration of the treated solution of step (3) is 1. mu.g/. mu.L;
the addition amount of the micrococcal nuclease in the step (3) is that the micrococcal nuclease and DNA are 1U: 20 mug;
the digestion temperature in the step (3) is 37 ℃;
the digestion time in the step (3) is 15 min.
4. A kit for chromatin fragmentation, comprising buffer a of claim 1 or claim 2, buffer CF1, buffer CF2, buffer N1, buffer N2 and a micrococcal nuclease.
5. Use of the method of any one of claims 1-3 and/or the kit of claim 4 for library construction and/or chromatin co-immunoprecipitation of ChIP-Seq.
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