CN108085379A - The ATAC-seq methods of calmodulin binding domain CaM positioning are opened applied to chromosome in tissue samples - Google Patents
The ATAC-seq methods of calmodulin binding domain CaM positioning are opened applied to chromosome in tissue samples Download PDFInfo
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- CN108085379A CN108085379A CN201711466019.5A CN201711466019A CN108085379A CN 108085379 A CN108085379 A CN 108085379A CN 201711466019 A CN201711466019 A CN 201711466019A CN 108085379 A CN108085379 A CN 108085379A
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Abstract
The present invention relates to the ATAC seq methods that calmodulin binding domain CaM positioning is opened applied to chromosome in tissue samples, are prepared including cell suspension, swivel base reacts and purifying, removal impurity, PCR amplification, high-flux sequence, Probability Detection.This method carries out DNA fragmentation using swivel base enzyme process, by cumbersome DNA fragmentation, end repair and connector coupled reaction and etc. become the simple enzymatic reaction of a step, shorten experimental period.The multidimensional information of regulation and control can be quickly obtained, the cell of about 3 to 5 orders of magnitude is saved than other methods.There is no the step of Piece Selection in experiment flow, can obtain the information such as the chromatinic position of open to the outside world, the binding site of transcription factor, the regulatory region of nucleosome and chromatin state simultaneously in this way.
Description
Technical field
Calmodulin binding domain CaM is opened the invention belongs to technical field of immunoassay more particularly to applied to chromosome in tissue samples
The ATAC-seq methods of positioning.
Background technology
In recent years, " big data " this vocabulary has become one of most common vocabulary instantly, and since last century 90
Age, bioinformatics, from initial DNA sequence analysis and protein sequence analysis, expand by development for many years
Open up the every field of biology so that the growth of biological data is surprising, and biology has also come into " big data " now
Epoch.
ATAC-seq technologies are made of ATAC experiments and high-flux sequence two parts, it is molecular biology research chromatin
The technology of accessibility.The technology was suggested for the first time in 2013, as MNase-seq, FAIRE-seq and DNase-seq
Replacement or compensation process.The key component of ATAC-seq experiments is effects of the transposase Tn5 to sample gene group DNA.ATAC-
Seq allows DNA and the connector for connecting particular sequence simultaneously that high efficiency cutting exposes using more active transposases of mutation.Separation
The DNA fragmentation of connector connection, by being used for high-flux sequence after PCR amplification.
The content of the invention
In view of this, what a kind of solution of present invention offer or part solved the above problems is applied to chromosome in tissue samples
The ATAC-seq methods of open calmodulin binding domain CaM positioning.
To achieve the effect that above-mentioned technical proposal, the technical scheme is that：It is opened applied to chromosome in tissue samples
The ATAC-seq methods of calmodulin binding domain CaM positioning are put, are comprised the following steps：
Step 1：It is prepared by cell suspension：
It prepares and contains 2 × 106~5 × 106Cell precipitation is homogenized at 4 DEG C in 500g and centrifuges by the cell precipitation of a cell
Mixture M is obtained after 10min, mixture A is obtained after mixture M is washed with the PBS buffer solution of precooling；PBS buffer solution includes：
200mM NaCl, 3mM KCl, 1mM MgCl2, 1mM CaCl2, 10mM Na2HPO4, 2mM KH2PO4, 0.1wt% Tween-20s,
The pH value of PBS buffer solution is 7.4；
Step 2：Swivel base reacts and purifying：
25ul TE buffer solutions, 2.5ul Tn5 transposases and 22.5ul DEPC processing water are added in mixture A, 37
Reaction 30min obtains mixture B in DEG C water-bath；TE buffer solutions include：10mM Tris-HCl, PH=8.0,1mM EDTA, PH=
8.0；
2.5ul ethidium bromide solutions are added in mixture B, after gently shaking up 15min, then are homogenized at 4 DEG C in 500g
Centrifugation 15min obtains mixture C；
Added in mixture C with the isometric chloroform alcoholic solution of mixture C, stand 10min after mild mixing, take upper strata
Clear liquid obtains mixture D；Chloroform alcoholic solution includes：Tris saturated phenols, chloroform, isoamyl alcohol, volume ratio 25:24:1；
It is staticly settled after adding in absolute ethyl alcohol in mixture D, obtains purifying DNA；The volume of mixture D and absolute ethyl alcohol
Than for 1:2；
Step 3：Remove impurity：
Washing buffer are added in DNA is purified, the Washing after rinsing is poured out after slightly rinsing 1~5min
Buffer obtains mixture E；
Blocking Solution are added in mixture E, the Blocking after standing is poured out after standing 0.5h
Solution obtains mixture F；
Antibody Solution are added in mixture F, the Antibody after standing is poured out after standing 0.5~1h
Solution obtains mixture G；
Washing buffer are added in mixture G, the Washing buffer after rinsing are poured out after rinsing 15min,
Obtain purified dna；
Step 4：PCR amplification：
It is expanded in advance using purified dna as template, the common 25ul of the reaction system expanded in advance, including 2ul purified dnas, 2ul draws
Object A, 0.2ul Taq archaeal dna polymerases, 1ul 10 × PCR of dNTPs, 2.5ul buffer, 17.3ul H2O；Mixing expands in advance
Reaction system, expanded in advance according to the following steps：First, 94 DEG C of denaturation 2min；Then, 30 are expanded according to following parameter
Xun Huan：94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 80sec；Finally, 72 DEG C of extension 5min；
It is expanded using the product expanded in advance as making choice property of template, the common 15ul of reaction system of selective amplification, including 2ul
The product expanded in advance, 1.2ul primers B, 0.12ul Taq archaeal dna polymerases, 0.6ul dNTPs, 1.5ul10 × PCR, 9.58ul
H2O；The reaction system of mixing selective amplification is expanded according to making choice property of the following steps：First, carried out according to following parameters
The first round expands：94 DEG C of 2min, 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 80sec；Then, successively decrease according to the circulating temperature of every wheel
1 DEG C, 10 wheel of amplification；Finally, then with 94 DEG C of 2min, 55 DEG C of 30sec, 72 DEG C of 80sec amplifications 30 obtain amplified production after taking turns；
Step 5：High-flux sequence：
Amplified production progress high-flux sequence is obtained into ATAC-seq data first；Then ATAC-seq data are connect
Read positioning is carried out again after head filtering, and obtained read is backfilling into reference gene group and obtains read backfill result；
Step 6：Probability Detection：
First, ATAC-seq data are read, and are compared in reference gene group, it is rich to search out open chromatin site
The characteristic peak of collection and the location information of peak maximum；The 100bp of both sides extension to the left and right, the number after extension are distinguished centered on peak maximum
In, the center of each DNA sequence dna is peak maximum；The sequence that DNA sequence dna is extracted and removed wherein repeatedly is obtained
The short sequences of DNA, and be numbered according to the secondary short sequences of ordered pair DNA of extraction, the number of the short sequences of DNA is continuous by 1
Positive integer sequence；
Then, the base quantity in the read quantity and the short sequences of each DNA in the short sequences of each DNA is counted；
Secondly, the open probability of the short sequences of DNA is calculated, is calculated with formula one：
Formula one：
Wherein, n is the number of the short sequences of DNA, is positive integer；I is the number of the short sequences of DNA, is positive integer；δ is DNA short
The open probability of sequence, δiIt is the open probability for the short sequences of DNA that number is i, δ and δiIt is no single for the real number between 0~1
Position；X is the read quantity in the short sequences of DNA, xiIt is the read quantity numbered in the short sequences of DNA for being i, x and xiFor positive integer；y
It is the base quantity in the short sequences of DNA, yiIt is the base quantity numbered in the short sequences of DNA for being i, y and yiFor positive integer；
Finally, δiProcessing method be：If δiMore than 0.5, then the short sequences of DNA that number is i are open chromatin positions
Point；If δiNo more than 0.5, the short sequences of DNA that number is i are not open chromatin sites.
The present invention useful achievement be：The present invention provides open calmodulin binding domain CaM positioning applied to chromosome in tissue samples
ATAC-seq methods, prepared including cell suspension, be swivel base reaction and purifying, removal impurity, PCR amplification, high-flux sequence, general
Rate detects.This method carries out DNA fragmentation using swivel base enzyme process, and cumbersome DNA fragmentation, end are repaired and connected instead with connector
Answer and etc. become the simple enzymatic reaction of a step, shorten experimental period.The multidimensional information of regulation and control can be quickly obtained, than it
His method will save the cell of about 3 to 5 orders of magnitude.There is no the step of Piece Selection in experiment flow, institute is in this way
The chromatinic position of open to the outside world, the binding site of transcription factor, the regulatory region of nucleosome and chromatin shape can be obtained simultaneously
The information such as state.
Specific embodiment
In order to which technical problems, technical solutions and advantages to be solved are more clearly understood, tie below
Embodiment is closed, the present invention will be described in detail.It should be noted that specific embodiment described herein is only explaining
The present invention is not intended to limit the present invention, and can be realized that the product of said function belongs to equivalent substitution and improvement, is all contained in this hair
Within bright protection domain.Specific method is as follows：
Embodiment 1：The present embodiment compared several open Chromatin domains identification technologies, as follows：
At present, the technology that can be used for carrying out open Chromatin domains identification has：ChIP-seq, DNase-seq, MNase-
Seq, FAIRE-seq, ATAC-seq, the fragmentation and enrichment mode of these types of technology are had nothing in common with each other.
(1)ChIP-seq：
The DNA sequence dna combined with transcription factor can be directly detected, but once sequencing can only provide the letter of a transcription factor
Breath.
(2)DNase-seq：
The substituted DNA sequence dna of nucleosome is preferentially cut using DNase I, the segment of generation is sequenced, is used for
The binding site of specific transcription factor is detected, but the technology, to the more demanding of cell initial amount, general cell quantity will reach
106~107, and the Preparatory work of experiment time is longer, it is necessary to 2-3 days.
(3)MNase-seq：
The technology just with DNase-seq technology complementations, mainly carries out fragmentation processing using restricted excision enzyme, until
It obtains until wrapping up or being transcribed the region of factor binding by nucleosome.
(4)FAIRE-seq：
DNA exposed in chromosome is fixed by organic solvent formaldehyde, is obtained afterwards by phenol chloroform naked
The DNA of dew, Preparatory work of experiment time are up to 3-4 days.
(5)ATAC-seq：
Can joint sequence be added in cutting DNA by Tn5 transposases, sequencing library can be obtained by PCR amplification,
It is more simple compared to DNase-seq steps, it is necessary to cell it is also less (102-105), can be completed in a few hours.By once
ATAC-seq, you can obtain chromosome all open Chromatin domains, not merely office under some specific space-time condition
It is limited to binding site or some specific acetylation of histone region of some transcription factor.
Embodiment 2：The present embodiment is specifically illustrated opens calmodulin binding domain CaM positioning applied to chromosome in tissue samples
The step of ATAC-seq methods, comprise the following steps：
Step 1：It is prepared by cell suspension：
It prepares and contains 2 × 106~5 × 106Cell precipitation is carried out following handle 3 times by the cell precipitation of a cell：4℃、
500g centrifuges 10min, removes supernatant liquor, is washed with the PBS buffer solution of precooling；PBS buffer solution includes：200mM NaCl, 3mM
KCl, 1mM MgCl2, 1mM CaCl2, 10mM Na2HPO4, 2mM KH2PO4, 0.1wt% Tween-20s；The pH value of PBS buffer solution
For 7.4, concentrated hydrochloric acid can be used to adjust pH value；
Step 2：Swivel base reacts and purifying：
Add in 25ul TE buffer solutions, 2.5ul Tn5 transposases and 22.5ul DEPC processing water, 37 DEG C of water-baths
30min；TE buffer solutions include：10mM Tris-HCl, PH=8.0,1mM EDTA PH=8.0；DEPC processing water can be selected
AM9920；
2.5ul ethidium bromide solutions are added in, gently shake up 15min, 4 DEG C, 500g centrifugations 15min；The ethidium bromide of selection
Solution 10mg/ml is dissolved in water；
Isometric chloroform alcoholic solution is added in, 10min is stood after mild mixing, takes supernatant liquor；Chloroform alcoholic solution into
Dividing includes：Tris saturated phenols, chloroform, isoamyl alcohol, volume ratio 25:24:1；
The absolute ethyl alcohol for adding in two volumes precipitates to obtain purifying DNA；
Step 3：Remove impurity：
Washing buffer are added in DNA is purified, slightly rinse 1~5min；
Washing buffer are poured out, add in Blocking Solution, stand 0.5h；
Blocking Solution are added, stand 0.5h；
Blocking Solution are poured out, add in Antibody Solution, stand 0.5~1h；
Antibody Solution are poured out, add in Washing buffer, Washing is poured out after rinsing 15min
Buffer obtains purified dna；
Step 4：PCR amplification：
It is expanded in advance using purified dna as template, the common 25ul of the reaction system expanded in advance, including 2ul purified dnas, 2ul draws
Object A, 0.2ul Taq archaeal dna polymerases (5U/ul), 1ul dNTPs (2.5uM), 2.5ul 10 × PCRbuffer, 17.3ul
H2O；The reaction system that mixing expands in advance, is expanded according to the following steps：First, 94 DEG C of denaturation 2min；Then, according to following
Parameter expands 30 Xun Huans：94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 80sec；Finally, 72 DEG C of extension 5min；Primer A is artificial
Two sections of oligonucleotide sequences of synthesis, it is necessary to according to template DNA sequence, that is, purified dna design complementary oligonucleotide chain as
Primer A, the corresponding different primer A of different cells；
It is expanded using the product expanded in advance as making choice property of template, the common 15ul of reaction system of selective amplification, including 2ul
The product expanded in advance, 1.2ul primers B, 0.12ul Taq archaeal dna polymerases (5U/ul), 0.6ul dNTPs (2.5uM), 1.5ul
10 × PCR, 9.58ul H2O；The reaction system of mixing selective amplification is expanded according to making choice property of the following steps：First,
First round amplification is carried out according to following parameters：94 DEG C of 2min, 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 80sec；Then, according to every
The circulating temperature of wheel is successively decreased 1 DEG C, 10 wheel of amplification；Finally, then with 94 DEG C of 2min, 55 DEG C of 30sec, 72 DEG C of 30 wheels of 80sec amplifications；Draw
Object B is two sections of artificial synthesized oligonucleotide sequences, it is necessary to which the product expanded in advance according to template DNA sequence, designs complementation
Oligonucleotide chain as primer B, the corresponding different primer B of different cells；
Step 5：High-flux sequence：
The product of step 4 is subjected to high-flux sequence with Hiseq PE150 first and obtains ATAC-seq data；Then will
ATAC-seq data carry out read positioning after carrying out connector filtering, and obtained read is backfilling into reference gene group and obtains read
Backfill result；
Step 6：Probability Detection：
First, the ATAC-seq data of sample are read, and are compared in reference gene group, search out open chromatin
The characteristic peak of site enrichment and the location information of peak maximum；Then, both sides are extended to the left and right respectively centered on peak maximum
100bp, in the data after extension, the center of each DNA sequence dna is peak maximum；Finally, DNA sequence dna is extracted and gone
Fall the sequence wherein repeated and obtain the short sequences of DNA, and according to the secondary short sequence numbers of ordered pair DNA of extraction, the number of the short sequences of DNA
For the continuous positive integer sequence by 1；
Then, the base quantity in the read quantity and the short sequences of each DNA in the short sequences of each DNA is counted；
Secondly, the open probability of the short sequences of DNA is calculated, is calculated with formula one：
Formula one：
Wherein, n is the number of the short sequences of DNA, is positive integer；I is the number of the short sequences of DNA, is positive integer；δ is DNA short
The open probability of sequence, δiIt is the open probability for the short sequences of DNA that number is i, δ and δiIt is no single for the real number between 0~1
Position；X is the read quantity in the short sequences of DNA, xiIt is the read quantity numbered in the short sequences of DNA for being i, x and xiFor positive integer；y
It is the base quantity in the short sequences of DNA, yiIt is the base quantity numbered in the short sequences of DNA for being i, y and yiFor positive integer；
Finally, δiProcessing method be：If δiMore than 0.5, then the short sequences of DNA that number is i are open chromatin positions
Point；Otherwise, then the short sequences of DNA that number is i are not open chromatin sites.
The present invention useful achievement be：The present invention provides open calmodulin binding domain CaM positioning applied to chromosome in tissue samples
ATAC-seq methods, prepared including cell suspension, be swivel base reaction and purifying, removal impurity, PCR amplification, high-flux sequence, general
Rate detects.This method carries out DNA fragmentation using swivel base enzyme process, and cumbersome DNA fragmentation, end are repaired and connected instead with connector
Answer and etc. become the simple enzymatic reaction of a step, shorten experimental period.The multidimensional information of regulation and control can be quickly obtained, than it
His method will save the cell of about 3 to 5 orders of magnitude.There is no the step of Piece Selection in experiment flow, institute is in this way
The chromatinic position of open to the outside world, the binding site of transcription factor, the regulatory region of nucleosome and chromatin shape can be obtained simultaneously
The information such as state.
The foregoing is merely the preferred embodiments of the invention, are not limited to claims of the invention.
Simultaneously it is described above, for those skilled in the technology concerned it would be appreciated that and implement, therefore other be based on institute of the present invention
The equivalent change that disclosure is completed, should be included in the covering scope of the claims.
Claims (1)
1. the ATAC-seq methods of calmodulin binding domain CaM positioning are opened applied to chromosome in tissue samples, which is characterized in that including with
Lower step：
Step 1：It is prepared by cell suspension：
It prepares and contains 2 × 106~5 × 106The cell precipitation is homogenized at 4 DEG C in 500g and centrifuges by the cell precipitation of a cell
Mixture M is obtained after 10min, mixture A is obtained after the mixture M is washed with the PBS buffer solution of precooling；The PBS delays
Fliud flushing includes：200mM NaCl, 3mM KCl, 1mM MgCl2, 1mM CaCl2, 10mM Na2HPO4, 2mM KH2PO4, 0.1wt%
Tween-20, the pH value of the PBS buffer solution is 7.4；
Step 2：Swivel base reacts and purifying：
25ul TE buffer solutions, 2.5ul Tn5 transposases and 22.5ul DEPC processing water are added in the mixture A, 37
Reaction 30min obtains mixture B in DEG C water-bath；The TE buffer solutions include：10mM Tris-HCl, PH=8.0,1mM EDTA,
PH=8.0；
2.5ul ethidium bromide solutions are added in the mixture B, after gently shaking up 15min, then are homogenized at 4 DEG C in 500g
Centrifugation 15min obtains mixture C；
Added in the mixture C with the isometric chloroform alcoholic solution of the mixture C, stand 10min after mild mixing, take
Supernatant liquor obtains mixture D；The chloroform alcoholic solution includes：Tris saturated phenols, chloroform, isoamyl alcohol, volume ratio 25:24:
1；
It is staticly settled after adding in absolute ethyl alcohol in the mixture D, obtains purifying DNA；The mixture D and the anhydrous second
The volume ratio of alcohol is 1:2；
Step 3：Remove impurity：
Washing buffer are added in the purifying DNA, the Washing after rinsing is poured out after slightly rinsing 1~5min
Buffer obtains mixture E；
Blocking Solution are added in the mixture E, the Blocking after standing is poured out after standing 0.5h
Solution obtains mixture F；
Antibody Solution are added in the mixture F, the Antibody after standing is poured out after standing 0.5~1h
Solution obtains mixture G；
The Washing buffer are added in the mixture G, the Washing after rinsing is poured out after rinsing 15min
Buffer obtains purified dna；
Step 4：PCR amplification：
It is expanded in advance using the purified dna as template, the common 25ul of reaction system of the pre- amplification, including pure described in 2ul
DNA, 2ul primer A, 0.2ul Taq archaeal dna polymerases, 1ul 10 × PCR of dNTPs, 2.5ul buffer, 17.3ul H2O；It is mixed
The reaction system of the even pre- amplification, is expanded in advance according to the following steps：First, 94 DEG C of denaturation 2min；Then, according to following
Parameter expands 30 Xun Huans：94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 80sec；Finally, 72 DEG C of extension 5min；
It is expanded using the product expanded in advance as making choice property of template, the common 15ul of reaction system of the selective amplification, including 2ul
The product of the pre- amplification, 1.2ul primers B, 0.12ul Taq archaeal dna polymerases, 0.6ul 10 × PCR of dNTPs, 1.5ul,
9.58ul H2O；The reaction system of selective amplification described in mixing is expanded according to making choice property of the following steps：First, according to
Following parameters carry out first round amplification：94 DEG C of 2min, 94 DEG C of 30sec, 56 DEG C of 30sec, 72 DEG C of 80sec；Then, according to every wheel
Circulating temperature is successively decreased 1 DEG C, 10 wheel of amplification；Finally, then with 94 DEG C of 2min, 55 DEG C of 30sec, 72 DEG C of 80sec amplifications 30 obtain after taking turns
Amplified production；
Step 5：High-flux sequence：
Amplified production progress high-flux sequence is obtained into ATAC-seq data first；Then by the ATAC-seq data into
Read positioning is carried out again after the filtering of row connector, and obtained read is backfilling into reference gene group and obtains read backfill result；
Step 6：Probability Detection：
First, the ATAC-seq data are read, and are compared in the reference gene group, search out open chromatin position
The characteristic peak of point enrichment and the location information of peak maximum；The 100bp of both sides extension to the left and right is distinguished centered on the peak maximum, is prolonged
In data after stretching, the center of each DNA sequence dna is the peak maximum；The DNA sequence dna is extracted and removes it
The sequence of middle repetition obtains the short sequences of DNA, and is numbered according to the short sequences of DNA described in the secondary ordered pair of extraction, the short sequences of DNA
Number is the continuous positive integer sequence by 1；
Then, the base quantity in the read quantity and the short sequences of each DNA in the short sequences of each DNA is counted；
Secondly, the open probability of the short sequences of DNA is calculated, is calculated with formula one：
Formula one：
Wherein, n is the number of the short sequences of DNA, is positive integer；I is the number of the short sequences of the DNA, is positive integer；δ is described
The open probability of the short sequences of DNA, δiIt is the open probability for the short sequences of DNA that number is i, the δ and the δiBetween 0~1
Real number, without unit；X is the read quantity in the short sequences of the DNA, xiIt is the read number numbered in the short sequences of DNA for being i
Amount, the x and the xiFor positive integer；Y is the base quantity in the short sequences of the DNA, yiIt is to number in the short sequences of DNA for being i
Base quantity, the y and the yiFor positive integer；
Finally, the δiProcessing method be：If the δiMore than 0.5, then the short sequences of DNA that number is i are the open dyes
Chromaticness site；If the δiNo more than 0.5, the short sequences of DNA that the number is i are not the open chromatin sites.
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Cited By (3)
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| CN111154835A (en) * | 2020-01-19 | 2020-05-15 | 广州基迪奥生物科技有限公司 | Method for constructing ATAC-seq sequencing library |
| WO2021027236A1 (en) * | 2019-08-12 | 2021-02-18 | 安诺优达基因科技(北京)有限公司 | Method for constructing dna library and application thereof |
| CN112410403A (en) * | 2020-11-04 | 2021-02-26 | 苏州京脉生物科技有限公司 | Preparation method and kit of ATAC-seq library with accurate quantification |
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| US20070141584A1 (en) * | 2005-12-20 | 2007-06-21 | Roberts Douglas N | Methods for assessment of native chromatin on microarrays |
| CN105339503A (en) * | 2013-05-23 | 2016-02-17 | 斯坦福大学托管董事会 | Transposition into native chromatin for personal epigenomics |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2021027236A1 (en) * | 2019-08-12 | 2021-02-18 | 安诺优达基因科技(北京)有限公司 | Method for constructing dna library and application thereof |
| CN111154835A (en) * | 2020-01-19 | 2020-05-15 | 广州基迪奥生物科技有限公司 | Method for constructing ATAC-seq sequencing library |
| CN112410403A (en) * | 2020-11-04 | 2021-02-26 | 苏州京脉生物科技有限公司 | Preparation method and kit of ATAC-seq library with accurate quantification |
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