CN108300774A - The improved open chromatin detection method of one kind and its kit and application - Google Patents
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Abstract
The invention discloses a kind of improved open chromatin detection methods, pulverize last addition PBS of cell tissue is resuspended, it centrifuges and removes supernatant, lysate is added into precipitation again, it is fully centrifuged after cracking and removes supernatant, precipitation is resuspended with PBS, and the sample after resuspension, which is crossed 40 μm of filter membranes, obtains the filtrate containing nucleus；Then the nucleus in filtrate is counted, 50,000 nucleus of picking centrifuges and remove supernatant in PBS, and swivel base system is added into precipitation, carries out swivel base reaction；Then the DNA of swivel base is purified, then carries out PCR amplification, finally PCR product is sequenced after purification.The method effectively can carry out chromatin Structure detection to clinical frozen tissue, its result and the result of flesh tissue inspection detection are completely the same, this method in combination with patient clinical data, to establish the model based on chromatin Structure detection and patient's prognosis, for promoting the development of precisely medical treatment, have great importance.
Description
Technical field
The invention belongs to biotechnologies.More particularly, to a kind of improved open chromatin detection method and its
Kit and application.
Background technology
In human cell's cell, DNA is combined to form chromatin with multi-layer structure with multiple protein.However, only
1% chromatin is active, is referred to as enlivening chromatin (open chromatin).These enliven chromatin, and ((such as promoter increases
Hadron) opening and closing and their positions and gene in nucleus expression regulation and cell biological behaviour
There is substantial connection.
All the time, researcher discloses active dyeing using DNase I digestions and high-flux sequence method (DNase-Seq)
Linear position of the matter in genome.But this method needs 107More than number of levels cell and a large amount of time are thrown
Enter, limits its application in clinical biopsy.2013 end of the year Stanford University Greenleaf taught and Howard Chang reports
ATAC-seq (the Assay for transposase-accessible chromatin using sequencing) technology in road
Appearance break through this limitation, provide completely new means to study chromatinic accessibility (ATAC-seq pass through Tn5 swivel bases
The adapters of sequencing is inserted into " accessible " region on genome to mark the region of regulation and control by enzyme, it is only necessary to 500~50000
A cell can obtain the chromatin open architecture collection of illustrative plates of high quality and as the intensification of sequencing depth (reaches 200millions
Reads), it might even be possible to be immediately seen transcription factor binding site (have the place that transcription factor combines not by Tn5 cleavages,
Trace (footprint) is formed, experiment flow and sample loss are enormously simplified.On July 12nd, 2017, ATAC-seq technologies hair
One of bright person Howard Chang are by tracking patient in histone deacetylase inhibitor (HDACi) cancer drug therapy process
The epigenetic state of middle Each point in time furthers investigate epigenetic genome and key transcription in time scale for the first time
The dynamic regulation mechanism of factor pair Patient drug's sensibility, and Accurate Prediction patient is to the sensibility of HDACi anticancer drugs, is new
Targeted therapy scheme provide foundation, also be research other diseases accurate medical treatment epigenetic regulation Mechanism establishing template.
In addition, being analyzed the DNA copy number of normal, mixing, host, tumour cell by ATAC-seq data, contaminated using the mankind
Colour solid figure shows difference of the copy number of human genome DNA in each cell type, and Howard Chang, which are confirmed, to be mixed
With multiple hereditary variations being reported in tumour cell, such as the increase of chr4q, chr8q and chr17q, chr10q and chr17p
Missing etc., and find that these variations are not happened in the host cell of normal person or patient.It is interesting that Howard
Chang has found that the DNA copy number difference of hereditary level can not reflect sensibility of the patient to drug, on the contrary, epigenetic layer
The transcription factor regulatory mechanism in face but can accurately predict that patient to the response situation of drug, illustrates apparent to patient again
The importance of gene group research has also prompted the limitation of conventional use of gene sequencing in current precisely medical research.
Regrettably, classical ATAC-seq technologies can only carry out the swivel base experiment of Tn5 in fresh cells, otherwise library
Quality it is very poor.Therefore applications of the ATAC-seq in clinical sample detection is seriously limited.In addition, classical ATAC-seq texts
There are more adaptor dimer and more than the large fragment of 600bp in library, the quality of sequencing is largely affected.
Invention content
The technical problem to be solved by the present invention is to overcome existing ATAC-seq technologies that can not be applied to clinical sample (frost
Tissue) and the libraries ATAC-seq in large fragment there are more adaptor dimer and more than 600bp can influence that matter is sequenced
The defect and deficiency of amount.A kind of improved ATAC-seq methods are provided, the method can effectively contaminate clinical frozen tissue
Chromatin structure detects, and result and the result of flesh tissue inspection detection are completely the same.This method can be effectively to clinical samples library
The chromatin Structure of tissue is detected, and combines the clinical data of patient, is detected and is suffered from based on chromatin Structure to establish
The model of person's prognosis has great importance for promoting the development of precisely medical treatment.
The object of the present invention is to provide a kind of improved open chromatin detection methods.
It is a further object of the present invention to provide one kind for opening chromatin detection kit.
The above-mentioned purpose of the present invention is to give realization by the following technical programs：
Present invention firstly provides a kind of nucleus extraction method, the cell tissue last addition PBS that pulverizes is resuspended, from
The heart simultaneously removes supernatant, then lysate is added into precipitation, fully centrifuges and remove supernatant after cracking, and precipitation is resuspended with PBS, will be weighed
Sample after outstanding crosses 40 μm of filter membranes and obtains the filtrate containing nucleus；The lysate includes that each group of following final concentration is grouped as：
50mM HEPES (pH 7.5), 140mMNaCl, 1mM EDTA (pH 8.0), 0.5%NP-40,0.25%TritonX-100.
The present inventor's early-stage study finds that classical nucleus extraction method is not suitable for frozen tissue, can not be from limited
Tissue in obtain cell check figure needed for follow-up study, and be difficult to carry out the swivel base experiment of Tn5, the Library Quality pole of preparation
Difference.For the present invention by optimizing the nucleus extraction method of cell tissue, especially independent development one kind can in frozen tissue
The method that high quality nucleus is obtained in (clinical sample)；With classical ATAC-seq first number take the fresh primary cell of certain amount or
Cell strain is compared by lysate cracking again after being counted, and the method for the invention is extracting nucleus from frozen tissue
Simultaneously, so that it may to carry out preliminary cracking to nucleus nuclear membrane, facilitate subsequent experiment.The nucleus extraction method of the present invention can
Cell check figure to extract a great deal of from limited frozen tissue can reach similarly horizontal with flesh tissue.
Preferably, the lysate also includes final concentration 10%glycerol；The present invention is on the basis of above-mentioned lysate
By the way that glycerol is further added, subsequent Library Quality can be made further to be promoted, by groping, find 10%
The best results of glycerol.
Preferably, the cell tissue is fresh cells tissue or frozen tissue.
Preferably, the mass volume ratio of the cell tissue and lysate is 5~10mg：1~2mL；The pass of ATAC-seq
Key is cracking, and cracking is insufficient, and Tn5 is not into going, and cracking too, and can destroy nuclear membrane, therefore to lysate and groups of cells
The ratio knitted has certain requirement.
Preferably, the mother liquid concentration of each component is in the lysate：1MHEPES (pH 7.5), 5MNaCl, 0.5MEDTA
(pH 8.0), 50%glycerol, 10%NP-40.
Preferably, in order to ensure the integrality and activity of nucleus in tissue, above-mentioned centrifugation be 4 DEG C of 2000g centrifugations 3~
5min；The PBS is the PBS of precooling.
The nucleus that the above method of the present invention obtains can meet the needs of open dyeing quality detection, therefore, above-mentioned nucleus
Application of the extracting method in open dyeing quality detection, the especially application in the open dyeing quality detection of frozen tissue also exist
In the scope of the present invention.
A kind of improved open chromatin detection method, to extract the thin of cell tissue using above-mentioned nucleus extraction method
Then karyon counts the nucleus in filtrate, 50,000 nucleus of picking centrifuges and remove supernatant, Xiang Chen in PBS
Swivel base system is added in shallow lake, carries out swivel base reaction.Then the DNA of swivel base is purified, then carries out PCR amplification, finally will
PCR product is sequenced after purification.
Specifically, described 50, the swivel base system of 000 nucleus is 25 μ L TDbuffer, 22.5 μ L ddH20,2.5 μ L
TD (Tn5 enzymes)；Wherein, cell check figure and the dosage of TD (Tn5 enzymes) are vital, they codetermine DNA fragmentation
Generate distribution.
Specifically, the swivel base is to be incubated 30 minutes for 37 DEG C in PCR instrument.
Specifically, the DNA to swivel base carries out purifying to be purified with AMPure beads：To swivel base
AMpure beads are added in DNA, are stood after mixing, then be placed in standing separation on Magneto separate frame, then removes supernatant and is used in combination
80% ethanol solution washs, and repeats the step, removes remaining ethanol solution, finally EB solution is used to elute.
Preferably, the volume ratio of DNA and the AMpure beads of the swivel base are 1：2~3.
Specifically, the PCR amplification methods of DNA after purification are identical as classical way.
Specifically, the PCR product purifying is to be purified with AMPure beads：It is added eventually into PCR reaction products
A concentration of 0.65 × AMpure beads, stand after mixing, then be placed in standing separation on Magneto separate frame, supernatant gone to newly
Guan Zhong, be added it is final concentration of 1.8 × AMpure beads, stand after mixing, then be placed in standing separation on Magneto separate frame, then
Removal supernatant is simultaneously washed with 80% ethanol solution, is repeated the step, is removed remaining ethanol solution, finally EB solution is used to elute.
Classical ATAC-seq carried out column purification by QiagenMinElute kit to the DNA of swivel base, by solidifying
Gel electrophoresis method is purified and is sorted to PCR product；Traditional mistake column purification and gel electrophoresis keep DNA losses more, can make
(it is seldom to obtain amount of DNA after 50000 cell DNA swivel bases) is reduced at library complexity, and the AMPure beads recycling of the present invention
The loss (about 30%) of DNA can be reduced；Remove the invalid sequencing segment in library：Classical ATAC-seq is not in library
Adaptor dimer and more than 600bp segments removal (retain all nucleosome pattern), however, if experiment
What is only focused on is open Chromatin domains rather than nucleosome location information, need not retain large fragment (the two generations survey of 600bp or more
Sequence instrument is extremely inefficient to the sequencing of 600bp or more segments).Therefore, we utilize AMPure beads, by the AMpure of two-wheeled
Beads screenings (by the ratio for controlling different AMPure beads and sample), eliminate adaptor dimer and are more than
The large fragment of 600bp substantially increases effective sequencing ratio of library DNA.
Meanwhile application of the above method in frozen tissue opens dyeing quality detection is also in the scope of the present invention.
A kind of kit for open dyeing quality detection, the kit includes above-mentioned lysate and AMPure
beads。
Preferably, also include 40 μm of cell strainer, Illumina Tagment DNA in the kit
Buffer, Illumina NexteraTagment DNA enzyme, NEBNext High-Fidelty 2xPCR Master
Mix, 100x SYBR Green I.
The kit of the present invention can carry out clinical frozen tissue open dyeing quality detection, result can reach and
Flesh tissue testing result is completely the same.
Compared with prior art, the invention has the advantages that：
The present invention provides a kind of improved ATAC-seq methods, pass through the nucleus extraction method progress to cell tissue
It improves, and substantially increases the quality in library by using AMpure beads technologies, the method can be effectively to clinical ice
Freeze tissue and carry out chromatin Structure detection, result and the result of flesh tissue inspection detection are completely the same.This method can be effectively
The chromatin Structure of clinical samples library tissue is detected, and combines the clinical data of patient, chromatin is based on to establish
The model of structure detection and patient's prognosis has great importance for promoting the development of precisely medical treatment.
Description of the drawings
Fig. 1 is PCR cycle number calculating method figure of the present invention.
Fig. 2 is to carry out screening front and back bioanalyzer analysis datagrams to library fragments using AMPure beads；On
Library (the text with the ATAC-seq of Nature Methods reports in 2013 without AMPure beads processing is shown in figure
Library is similar, the nucleosome distribution of expression characteristics), figure below is the library by AMPure beads processing.
Fig. 3 is the pearson related coefficients of frozen liver tissues and fresh liver tissue ATAC-seq.
Fig. 4 is frozen liver tissues and fresh liver tissue UCSC genome browser sectional drawings.
Fig. 5 is the nucleus lysate optimization process figure of the present invention.
Fig. 6 is the comparison diagram of the method for the present invention and the method for Nature Methods reports in 2013.
Specific implementation mode
It is further illustrated the present invention below in conjunction with Figure of description and specific embodiment, but embodiment is not to the present invention
It limits in any form.Unless stated otherwise, the present invention uses reagent, method and apparatus routinely try for the art
Agent, method and apparatus.
Unless stated otherwise, following embodiment agents useful for same and material are purchased in market.
The opening chromatin detection method of 1 frozen liver tissues of embodiment and fresh liver tissue
One, method
1, under liquid nitrogen, 5mg fresh liver tissues and 5mg are freezed hepatic tissue grind into powder respectively using grinding ware, so
After be transferred in 1.5mLEP pipes；
2, the PBS of 1mL 1x precoolings is added into tissue powder, 4 DEG C of 2000g centrifuge 3min after resuspension；3, after removing supernatant
1mL LB1 lysates are added into pellet to be resuspended；Then 10min is shaken at 4 DEG C；The preparation of the lysate is as shown in table 1：
Table 1
4 and then sample is transferred in 2mL glass homogenizers, is softly homogenized under 15, is then transferred into 1.5mLEP pipes
In, 4 DEG C of 2000g centrifuge 5min；
5, it draws supernatant and pellet is resuspended with the PBS of 1mL 1x precoolings；
6, the cellstrainer (being placed in 6 orifice plates) of 40 μm of filter membranes of the sample after resuspension is filtered；
7, nucleus is counted with tryptanblue；
8,50,000 nucleus are transferred in 1.5mL EP, liquid are supplied to 50 μ L using PBS, then 4 DEG C of 800g
Centrifugation 10 minutes；
9, it after removing supernatant carries out that 25 μ L TDbuffer, 22.5 μ L ddH20,2.5TDE (Tn5 enzymes) are added, and is transferred to
In PCR pipe, it is incubated 30 minutes for 37 DEG C in PCR instrument；
10, purified to the DNA of swivel base with AMPurebeads：
(1) 100 μ LAMpurebeads are added, blows and beats 10 times, is stored at room temperature 15min；
(2) and then beads it is placed on magneticrack, is stored at room temperature 2min；
(3) supernatant is removed；
(4) ethyl alcohol of 200 μ L, 80% Fresh is added；
(5) supernatant is removed after being incubated at room temperature 30sec；
(6) it repeats to be cleaned once with ethyl alcohol；
(7) after removing supernatant, of short duration spindown is carried out, then EP pipes are put back into magneticrack, removal is residual
Remaining ethyl alcohol；
(8) room temperature air-dries 5min；
(9) EP pipes are moved away from magneticrack；
(10) beads is resuspended with 16.5 μ LEBbuffer；
(11) it is incubated at room temperature 2min；
(12) EP pipes are put into magneticrack, carefully draw 15 μ L supernatants；
11, PCR amplification is carried out to purified swivel base DNA, the PCR is shown in reaction system such as table 2, PCR programs such as table 3
It is shown：
Table 2
Table 3
| Component | Volume |
| Swivel base DNA | 15μL |
| Nextera PCR primer index N7XX (25 μM) | 2.5μL |
| Nextera PCR primer index S5XX (25 μM) | 2.5μL |
| NEBNext High-Fidelity 2x PCR Master | 25μL |
| 100x SYBR Green I** | 0.3μL |
| H20 | 4.7μL |
| Total volume | 50μL |
12, in order to reduce GC and DNA fragmentation size bias, the pre- PCR reactions of following one are carried out, to determine remaining sample
PCR cycle number (avoiding supersaturation)：Wherein, reaction system is as shown in table 4, and PCR programs are shown in table 5；
Table 4
Table 5
13, the optimum cycle number (as shown in Figure 1) of 45 μ LPCR reactions of residue is calculated：
Method：The recurring number corresponding to 1/4 maximum fluorescence intensity of each curve is measured, which is then remaining 45 μ L samples
The optimum cycle number of this reaction；
14, PCR amplification (in addition to recurring number changes, remaining program is as 12) is carried out to remaining 45 μ L.
15, PCR product is carried out carrying out two-wheeled AMPurebeads purifying；
The first round (0.65XAMPure beads)
(1) 45 μ LPCR reaction products are supplied with water to 50 μ L, 32.5 μ LAMPurebeads is added, blow and beat 10 times；
(2) it is stored at room temperature 15min；
(3) and then beads it allows on magneticrack,
(4) 2min is stood；
(5) supernatant is transferred in a new EP pipe；
Second wheel (1.8XAMPure beads)
(1) 57.5 μ LAMpurebeads are added into supernatant, blows and beats 10 times, is stored at room temperature 15min；(2) beads is put
On magneticrack, it is stored at room temperature 2min；
(3) supernatant is removed；
(4) ethyl alcohol of 200 μ L, 80% fresh configurations is added；
(5) supernatant is removed after being incubated at room temperature 30sec；
(6) it repeats to be cleaned once with ethyl alcohol；
(7) after removing supernatant, of short duration spindown is carried out, then EP pipes are put back into magneticrack, removal is residual
Remaining ethyl alcohol；
(8) room temperature air-dries 5min；
(9) EP pipes are moved away from magneticrack；
(10) beads is resuspended with 22.5 μ LEBbuffer, is incubated at room temperature 2min；
(11) beads is put into magneticrack, carefully draws 20 μ L supernatants.
16, PCR product after purification is sequenced.
Two, result
1, frozen liver tissues library of the invention is after using two-wheeled AMPure beads to handle, in library
Adaptor dimer (the red frame in the left side) and more than the segment of 600bp (the red frame in the right) by well remove (as shown in Figure 2).And
Without AMPure beads processing library (it is similar with the library of ATAC-seq of Nature Methods reports in 2013, be in
Existing characteristic nucleosome distribution).
2, the libraries ATAC-seq of the present embodiment fresh liver tissue and frozen liver tissues carry out (after AMPure processing)
Sequencing, as a result, it has been found that the two generates about 72423 peaks；The pearson related coefficients of the two are up to 0.995 (such as Fig. 3 institutes
Show)；UCSC genome browsers show fresh (such as Fig. 4 similar with the ATAC-seq testing result height of the liver organization of frost
It is shown), the methods and results for repeating the experiment display present invention are stable, reliable.
The optimization of 2 lysate of embodiment
The key of lysate is to introduce Triton to be cracked (Nature in 2013 in the embodiment of the present invention 1
Methods articles be used only NP-40), the step for so that the quality in library is significantly improved；In addition, after we introduce glycerol
(quality of a concentration of 50%), library is further enhanced；Again by groping the various concentration of glycerol, 10% is found
The best results of glycerol；The results are shown in Figure 6 for it.
The method of the classics of comparative example 1 carries out the open dyeing quality detection of frozen liver tissues
Using classical method (protocol for being published in Nature Methods in 2013) to fresh and frost small
Mouse liver organization carries out ATAC-seq experiments, some special peak, but background can be presented in as a result fresh liver organization
(noise) higher, and background is high in frozen tissue, can not see special peak at all.(figure below is UCSC
Region near genome browser interception house keeper's Gene A CTB genes).Therefore it can not be freezed with classical method
The ATAC-seq of tissue is tested.And the method that we independently improve without wheel fresh or result in frozen liver tissues all
Special peak is presented and almost without background (Fig. 5), shows further the superiority of the method for the present invention.
Claims (10)
1. a kind of nucleus extraction method, which is characterized in that pulverize last addition PBS of cell tissue is resuspended, centrifugation is simultaneously
Supernatant is removed, then lysate is added into precipitation, fully centrifuges and remove supernatant after cracking, precipitation is resuspended with PBS, after resuspension
Sample cross 40 μm of filter membranes and obtain the filtrate containing nucleus；
The lysate includes each component of following final concentration：50mM HEPES（pH 7.5）, 140 mMNaCl, 1 mM EDTA
（pH 8.0）, 0.5% NP-40,0.25% TritonX-100.
2. according to the method described in claim 1, it is characterized in that, the lysate also includes 10% glycerol of final concentration.
3. according to the method described in claim 1, it is characterized in that, the mass volume ratio of the cell tissue and lysate is 5
~10 mg：1~2mL.
4. application of any one of claims 1 to 3 the method in open dyeing quality detection.
5. a kind of improved open chromatin detection method, which is characterized in that extracted using 2 the method for claims 1 or 2 thin
The nucleus of born of the same parents' tissue, then counts the nucleus in filtrate, 50,000 nucleus of picking is centrifuged and gone in PBS
Except supernatant, swivel base system is added into precipitation, carries out swivel base reaction；Then the DNA of swivel base is purified, then carries out PCR
Amplification, finally PCR product is sequenced after purification.
6. according to the method described in claim 5, it is characterized in that, the DNA to swivel base carries out purifying to use AMPure
Beads is purified：AMpure beads are added into the DNA of swivel base, are stood after mixing, then be placed on Magneto separate frame and stand
Then separation removes supernatant and is washed with 80% ethanol solution, repeats the step, remove remaining ethanol solution, finally use EB molten
Liquid elutes.
7. according to the method described in claim 6, it is characterized in that, the volume of DNA and the AMpure beads of the swivel base
Than being 1：2~3.
8. according to the method described in claim 5, it is characterized in that, PCR product purifying is to be carried out with AMPure beads
Purifying：Be added final concentration of 0.65 into PCR reaction products × AMpure beads, stand after mixing, then be placed in Magneto separate
Standing separation on frame goes to supernatant in new pipe, be added it is final concentration of 1.8 × AMpure beads, stand after mixing, then
It is placed in standing separation on Magneto separate frame, then remove supernatant and is washed with 80% ethanol solution, the step is repeated, removal is remaining
Ethanol solution finally uses EB solution to elute.
9. application of any one of claim 5~8 the method in frozen tissue opens dyeing quality detection.
10. a kind of kit for open dyeing quality detection, which is characterized in that include cracking as claimed in claim 1 or 2
Liquid.
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| CN111705128A (en) * | 2020-04-03 | 2020-09-25 | 苏州睿迈英基因检测科技有限公司 | Improved method for detecting breast cancer frozen tissue open chromatin |
| CN111705111A (en) * | 2020-04-03 | 2020-09-25 | 苏州睿迈英基因检测科技有限公司 | Improved method for detecting gastric cancer frozen tissue open chromatin |
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