CN106591955B - Construct high-resolution, the method in the unicellular library Hi-C of large information capacity - Google Patents
Construct high-resolution, the method in the unicellular library Hi-C of large information capacity Download PDFInfo
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Abstract
The present invention provides a kind of building high-resolution, the method in the unicellular library Hi-C of large information capacity.This method original samples amount is few, high resolution, contains much information and easy to operate.The method in the building library Hi-C of the invention includes the following steps: to obtain a small amount of chromatin being immobilized；The chromatin being immobilized obtained in the step B is digested, the chromatin fragments being immobilized；The chromatin fragments being immobilized obtained in the step C are directly reconnected, the chromatin fragments being immobilized reconnected；The chromatin fragments of reconnect obtained in the step D being immobilized are made to release immobilization, released dna segment；The DNA fragmentation discharged in the step E is expanded, amplified production is obtained；And using the amplified production as DNA fragmentation to be sequenced, sequencing DNA library is constructed.
Description
Technical field
The present invention relates to the library construction sides that one kind can capture chromatin three-dimensional conformation within the scope of full-length genome
Method belongs to gene sequencing technology field.
Background technique
DNA is the carrier of cytogenetics information, is present in the form of chromatinic in each cell in vivo, and control
Make the process of entire vital movement.The current overwhelming majority is with the sequence of base in researching DNA molecule for the research of DNA information
(one-dimension information of DNA) Lai Jinhang is arranged, the rule of vital movement is probed by analysis base arrangement information.
Nucleus in time of day is a narrow three-dimensional space, and the DNA of straight-chain molecular structure can be with complexity
Rolled fashion be located in nucleus, former one-dimensional DNA sequence dna is endowed three-dimensional conformation, and results in the gene of large amount of complex
Regulating and controlling effect mode.In this regard, simple one-dimensional DNA sequence dna information be due to that cannot provide true DNA spatial distribution relevant information,
Therefore also without method interpretation, the series of genes as caused by space conformation regulates and controls phenomenon.
To solve this problem, has a series of detection method at present.Such as 3c (chromosome structure capture) method and spread out
Raw 4c, 5c method.These methods form DNA structure using cell nuclear protein and fix with sequencing for basic detection means
The factor DNA sequence dna that buildings have spatial structural form such as joins by the segment to DNA later again, finally uses sequencing technologies
Chromatin dna information is detected, and calculates it and is distributed and interacts in space.Although such method to a certain extent can
Chromatin interaction information in part is enough provided.But due to its method and technical restriction, this kind of methods is only capable of detection fixed point
Or partial DNA interaction sites, the three-dimensional interaction information on full nuclear level can not be probed into.Therefore inevitably big
Amount information will omit.In the discovery for unknown interaction information, this point is particularly important.
Recently as the appearance of high throughput sequencing technologies, the acquisition of extensive genomic information becomes to be more easier.Hi-
C technology is the method in conjunction with high-flux sequence, the technology detected to chromatinic information in entire nucleus.Hi-C skill
Art is a kind of deriving technology of chromosomal conformation capture (Chromosome conformation capture, referred to as 3C), is
Refer to based on high-flux sequence carry out chromosomal conformation capture, it can be captured within the scope of full-length genome different genes seat it
Between spatial interaction, study three-dimensional space in controlling gene DNA element.
For example, patent document 1 and non-patent literature 1 have reported a kind of Hi-C method, this method utilizes the fixed dyeing of formaldehyde
Then matter structure interrupts protogene group sequence by restriction enzyme, and after carrying out biotin labeling, re-attached is formed
New DNA molecular with structural information.In this course, if the DNA molecular segment connection of two different genes group positions
A hybrid molecule is formed, this will be considered as the two DNA moleculars spatially evidence adjacent to one another.Then to DNA into
Row purifying is simultaneously smashed, then fish for the biotin molecule marked and be taken, the DNA spatially to interact needed for enrichment acquisition
Hybrid molecule.It finally constructs the library of high-flux sequence and carries out both-end sequencing detection, obtain the phase of full dyeing matter spatially
Interaction information.1) this method mainly includes the following steps: first to 106It is solid that a above cell sample carries out formaldehyde crosslinking
Fixed, the DNA for being closer its inner space passes through protein-crosslinking together, and collects cell；2) using cracking system and match
It closes grinding and carries out cell cracking, obtain the nucleus of separation；3) using restricted type restriction endonuclease (such as EcoR I) to cell after crosslinking
Chromatin carry out digestion；4) biotin is marked to digestion end, and forms flat end；5) using DNA ligase to flat
End is attached, and will have biggish probability to connect to form new molecule between the DNA fragmentation on the same corsslinking molecular；6)
High temperature (65 temperature) processing reverse cross-link, discharges double chain DNA molecule；7) the not connected terminal biotin label of removal；8) to DNA into
Row fragmentation, and carry out biotin specificity and transfer, it is enriched with hybrid molecule connection site region；And 9) building Illumina is surveyed
Preface library carries out both-end sequencing, obtains data.
But this method is with 106Great amount of samples more than a cellular level originates, and has used in whole flow process large-scale
Grinding and extracting method, are not particularly suited for a small amount of samples (105Below a cell), particularly individual cell level Hi-C detection.
Further, since this method is directed to a large amount of cell samples, the total chromatin conformation of group's cell can only be detected, it can not be to cell dyeing
Texture is as the difference between individual is detected and is compared.
On the other hand, non-patent literature 2 has reported a kind of slender karyon Hi-C method, but this method original samples are still
106A large amount of cell samples of the order of magnitude.Then pre-processing is beaten using the fixed chromatin Structure of formaldehyde by restriction enzyme
Disconnected protogene group sequence, and after carrying out biotin labeling, re-attached forms the new DNA molecular for having structural information.At this
During one, if the DNA molecular segment of two different genes group positions connects to form a hybrid molecule, this will be considered as
The two DNA moleculars spatially evidence adjacent to one another.Next, being carried out under the microscope to the nucleus Jing Guo pre-treatment
It selects, obtains single nucleus, then carry out for individual cells core sample that crosslinking removal, biotin fishing takes and restriction enzyme
Enzyme carries out fragmentation to DNA molecular, and enrichment obtains the required DNA hybrid molecule spatially to interact.Finally construct high pass
It measures the library of sequence and carries out both-end sequencing detection, obtain the interaction information of full dyeing matter spatially.This method is main
Include the following steps: 1) first to 106A above cell sample carries out formaldehyde crosslinking and fixes, and is closer its inner space
DNA by protein-crosslinking together, and collect cell；2) using cracking system and cooperate grinding carry out cell cracking, obtain
Isolated nucleus；3) digestion is carried out using the chromatin of restricted type restriction endonuclease (such as EcoR I) to cell after crosslinking；4) to digestion
End carries out biotin labeling, and forms flat end；5) flat end is attached using DNA ligase, is in the same friendship
There to be biggish probability to connect to form new molecule between DNA fragmentation on connection molecule；6) slender karyon is carried out under the microscope
Picking obtains the slender karyon sample by Hi-Cization processing；7) high temperature is carried out to slender karyon sample and removes formaldehyde crosslinking, released
Put DNA；8) biotin specificity is carried out to DNA molecular to transfer, be enriched with hybrid molecule connection site region；9) using second of limit
Property restriction endonuclease processed carries out fragmentation to DNA, adapts it to Illumina sequencing library Insert Fragment size；10) to combine life
The magnetic bead of object element labeled fragment (hybrid molecule for the segment that spatially interacts) is carrier, constructs Illumina sequencing library,
Both-end sequencing is carried out, data are obtained.
Although the method goes crosslinking and library construction stage to start to be operated for slender karyon the later period, and most
The result obtained eventually is the Hi-C of individual cell level as a result, still at processing initial stage, due to needing the formaldehyde by large volume to hand over
The means such as connection processing and grinding smudge cells obtain nucleus, therefore this method still needs 106A large amount of samples more than a cell
This conduct starting carries out Hi-Cization processing, therefore does not realize that the Hi-C for unicellular sample base level is detected really, still
A small amount of samples (10 can not be so suitable for5Below a cell), particularly unicellular sample.Furthermore since this method expands in library
Digestion, end reparation, biotin labeling, blunt end cloning, biotin is undergone to transfer before increasing, the first-class multiple steps of adjunction,
And these steps are operated in the case where a set of genome copies, the efficiency of each step can all influence to capture to the end
Information content, cause information loss in whole experiment process serious, the DNA interaction segment number finally captured is also very low.This
Outside, chromatin digestion and reconnection product fragmentation are carried out using two kinds of restriction nuclease enzymes due to this method respectively.In order to guarantee
The efficiency of reconnection product fragmentation, the frequency that restriction endonuclease used in the step occurs in genome will digest institute much higher than chromatin
The frequency occurred with restriction endonuclease.The four base enzymes that this reason causes this method not to be available the appearance of genome high frequency carry out
It is lower to eventually lead to gained Hi-C literature data resolution ratio for chromatin digestion.It also attempted to use genome in non-patent literature 2
The four base enzymes that medium-high frequency occurs carry out chromatin digestion, but due to subsequent restriction endonuclease of the reconnection product fragmentation without higher frequency
It can use, resolution ratio and information content can not be improved.Moreover, this method is the slender karyon of picking to the unicellular scheme studied,
Its operation difficulty and requirement to instrument, technology are unicellular much higher than picking.
Therefore, above-mentioned Hi-C method in the prior art be not can be suitable for it is unicellular or a small amount of cell, practical
The Hi-C method of change.
Patent document 1
International publication number WO2010036323A1 non-patent literature 1
Lieberman-Aiden E et al.Comprehensive mapping of long-range
interactions reveals folding principles of the human genome.Science 326,289-
293(2009)
Non-patent literature 2
Takashi Nagano et al.Single-cell Hi-C reveals cell-to-cell
variability in chromosome structure.Nature 502,59-64(2013)
Summary of the invention
In view of above-mentioned the deficiencies in the prior art, it is an object of the invention to: provide one kind can be suitable for it is a small amount of
The method in the building library Hi-C of chromatin (the even single celled chromatin of a small amount of cell).
The present inventor has made intensive studies to solve above-mentioned technical problem, in traditional method for constructing the library Hi-C
On the basis of cleverly improved, initiated and be suitable for a small amount of chromatinic Hi-C library constructing method, so as to complete this
Invention.
That is, the present invention includes:
1. a kind of method for constructing the library Hi-C, this method include the following steps:
Step B: a small amount of chromatin being immobilized is obtained；
Step C: the chromatin being immobilized obtained in the step B is digested, the dyeing being immobilized
Matter segment；
Step D: the chromatin fragments being immobilized obtained in the step C are directly reconnected, and obtain weight
The chromatin fragments being immobilized newly connected；
Step E: so that the chromatin fragments of reconnect obtained in the step D being immobilized is released immobilization, release
Put DNA fragmentation；
Step F: the DNA fragmentation discharged in the step E is expanded, amplified production is obtained；And
Step H: using the amplified production as DNA fragmentation to be sequenced, sequencing DNA library is constructed.
2. according to method described in item 1, wherein a small amount of chromatin being immobilized is 10-6~102The dyeing of ng
Matter, in terms of naked DNA.
3. the method according to item 1 or 2, wherein a small amount of chromatin being immobilized is 10-5The dye of~10ng
Chromaticness, in terms of naked DNA.
4. the method according to any one of item 1~3, wherein using deoxyribonuclease to institute in the step C
The chromatin being immobilized is stated to be digested.
5. according to method described in item 4, wherein the deoxyribonuclease is I type restriction enzyme, the limitation of II type
Property restriction endonuclease or III type restriction enzyme.
6. the method according to any one of item 1~5, wherein using viscous end or blunt end cloning in the step D
Method reconnects the chromatin fragments being immobilized obtained in the step C.
7. the method according to any one of item 1~6, further include:
Step G: by amplified production fragmentation obtained in the step F, smaller DNA fragmentation is obtained；And
In the step H, using smaller DNA fragmentation obtained in the step G as DNA fragmentation to be sequenced, building is surveyed
Sequence DNA library.
8. according to method described in item 7, wherein interrupt method, swivel base enzyme process, restriction endonuclease enzyme using ultrasound in the step G
Cutting method or hydraulic shear cutting method are by the amplified production fragmentation.
9. according to method described in item 8, wherein the size of smaller DNA fragmentation obtained in the step G be 50~
1000bp。
10. the method according to any one of item 1~9, further include:
Step A: the cell that a small amount of chromatin is immobilized is obtained；And
In the step B, the cell obtained in the step A is cracked, obtains a small amount of chromatin being immobilized.
11. according to method described in item 10, wherein the cell that a small amount of chromatin is immobilized is 1~10000
Cell.
12. according to method described in item 10, wherein the cell that a small amount of chromatin is immobilized is 1~1000
Cell.
13. according to method described in item 10, wherein the cell that a small amount of chromatin is immobilized is individual cells.
14. according to method described in item 10, wherein the step A includes:
Step A-1: immobilizing the chromatin of a certain amount of cell, obtains what a certain amount of chromatin was immobilized
Cell；And
Step A-2: a small amount of dye of picking in the cell that a certain amount of chromatin obtained from the step A-1 is immobilized
The cell that chromaticness is immobilized.
15. according to method described in item 10, wherein the step A includes:
Step A-3: immobilizing the chromatin of a small amount of cell, obtain a small amount of chromatin be immobilized it is thin
Born of the same parents.
16. a kind of method for the Chromatin domains that measurement may spatially interact, this method comprises:
The library Hi-C is constructed using method described in any one of item 1~15；And
The all or part in the library Hi-C is sequenced, and by obtained information and chromatin dna primary sequence
Information is compared.
Invention effect
According to the present invention, provide that a kind of original samples amount is few, high resolution, contains much information, constructs Hi- easily to operate
The method in the library C.
Aiming at the problem that prior art can not handle trace sample and unicellular sample, the present invention from most originate the step of just
It can be operated for single cell or trace sample (1-1000 cell), solve small sample amount cell and carry out Hi-C detection
Problem.
Aiming at the problem that prior art can not detect unicellular chromatin conformation, the present invention can be carried out for individual cells
Hi-C analysis, the difference of the chromatin conformation of detection and research individual cells and different iuntercellular chromatin conformations.
For the problem that the interaction of DNA in the prior art information loss is larger, the present invention has abandoned the end after digestion
It repairs, biotin labeling and transfer, and connection effect can be replaced using the higher viscous end connection of joint efficiency
The lower blunt end cloning of rate, thus the loss of information content is reduced, more chromatin conformation information can be captured.
The lower problem of chromatin conformation resolution ratio is obtained for the prior art, the present invention can be in library fragments Shi Caiyong
The method interrupted at random, fragmentation effect are much higher than digestion with restriction enzyme.This operation can when chromatin is digested
Restriction enzyme of the restriction enzyme of higher resolution as identified 4 base sequences is selected, so as to improve chromatin
Conformation resolution ratio.
Aiming at the problem that the slender karyon operating difficulties of picking in the prior art, the present invention can carry out text with picking individual cells
Library building, difficulty are less than picking individual cells core.
The specific embodiment of invention
The scientific and technical terminology referred in this specification has meaning identical with the normally understood meaning of those skilled in the art,
If any conflict, the definition in this specification shall prevail.
In an aspect, the present invention provides a kind of method (method of the invention) for constructing the library Hi-C, this method packet
Include following step:
Step B: a small amount of chromatin being immobilized is obtained；
Step C: the chromatin being immobilized obtained in the step B is digested, the dyeing being immobilized
Matter segment；
Step D: the chromatin fragments being immobilized obtained in the step C are directly reconnected, and obtain weight
The chromatin fragments being immobilized newly connected；
Step E: so that the chromatin fragments of reconnect obtained in the step D being immobilized is released immobilization, release
Put DNA fragmentation；
Step F: the DNA fragmentation discharged in the step E is expanded, amplified production is obtained；And
Step H: using the amplified production as DNA fragmentation to be sequenced, sequencing DNA library is constructed.
Preferably, method of the invention further include:
Step G: by amplified production fragmentation obtained in the step F, smaller DNA fragmentation is obtained；And the step H
In, using smaller DNA fragmentation obtained in the step G as DNA fragmentation to be sequenced, construct sequencing DNA library.
Method of the invention further include:
Step A: the cell that a small amount of chromatin is immobilized is obtained；And in the step B, crack in the step A
The cell of acquisition obtains a small amount of chromatin being immobilized.
Mode for obtaining the cell that a small amount of chromatin is immobilized in step A is not particularly limited, for example, can be with
The chromatin of a certain amount of cell is immobilized first, obtains the cell that a certain amount of chromatin is immobilized, then from
The cell that a small amount of chromatin of picking is immobilized in the cell that obtained a certain amount of chromatin is immobilized；Or it can be straight
It connects and the chromatin of a small amount of cell is immobilized, obtain the cell that a small amount of chromatin is immobilized.It should be noted that
Here cell can also be nucleus.
In the present specification, Hi-C refers to chromatin three-dimensional space interaction group, it is that one kind can be in full-length genome range
Chromatinic three-dimensional structure and different region of DNA domains spatially correlation are studied in interior progress chromatin space conformation capture
Method.The library Hi-C refers to: obtaining possible chromatin interaction information in Hi-C method by high-flux sequence, is used for
The DNA library of this high-flux sequence is the library Hi-C.
In the present specification, " a small amount of chromatin " refers to the amount that the Hi-C method of the as little as prior art can not be operated
Chromatin, can usually refer to the chromatin of 1~10000 cell perhaps the chromatin of 1~1000 cell or 1~
The chromatin of 100 cells, the chromatin of even 1 cell (unicellular), then the dyeing of even 1 cell (unicellular)
A part of matter.If in mass, described " a small amount of chromatin " can be 10-6~102Ng, preferably 10-5~10ng, with
Naked DNA meter.
In the present specification, " a small amount of cell " refers to the quantity that the Hi-C method of the as little as prior art can not be operated
Cell, can usually refer to 1~10000 cell perhaps 1~1000 cell or 1~100 cell, even 1
Cell (unicellular).
In the present specification, " immobilization " refers to: in cell, chromatin part close to each other on three-dimensional space by with
State close to native conformation is fixed.In the present specification, chromatin also includes chromosome morphology.The immobilization usually may be used
By the way that the protein cross on chromatin is carried out.The cross-linking method of protein on chromatin is those skilled in the art
It is known, for example, can be used alone ultraviolet light, or can be used alone tetranitromethane, carbon imidodicarbonic diamide class, formaldehyde,
If methanol, valeral, mustargen, dimethyl sulfate, formaldehyde releaser, acid imide esters, has Mitomycin C, mustard gas and sweeps ethyl alcohol
The chemical reagent such as logical sequence can also be crosslinked using the method for above chemical reagent combination ultraviolet light irradiation.For example, passing through first
Cell in the case where carrying out the immobilization, can be placed in water, the TE of appropriate amount (such as 1~10000000 μ L) by aldehyde crosslinking
Cell suspending liquid drop is made in buffer, physiological saline, PBS or cell culture medium, adds appropriate amount (such as 1-10000000
μ L) formalin (to its concentration there is no limit, such as can be 1~20 weight %), be stored at room temperature certain time (such as 1-
100min), it is crosslinked.Then, a certain amount of amino acid (a kind of amino acid or a variety of amino is added in Xiang Shangshu reaction solution drop
The mixture of acid) or protein (such as BSA etc.) terminate cross-linking reaction.
It, can be first to a certain amount of (such as 10 in the step A5More than a, preferably 106~108It is a) cell dyeing
Matter immobilizes, and obtains the cell that a certain amount of chromatin is immobilized, and then therefrom a small amount of chromatin of picking is consolidated again
Surely the cell changed；Can also the chromatin directly to a small amount of cell immobilize, consolidated to obtain a small amount of chromatin
Surely the cell changed.The picking of a small amount of cell can pass through capillary tube method, dilution method, gradient dilution method or flow cytometer point
Method is selected to carry out.
In the step B, the cell obtained in the step A is cracked, thus the dyeing being immobilized
Matter.Cell cracking can usually be carried out by the way that the cell to be placed in cell pyrolysis liquid appropriate.The cell pyrolysis liquid
Formula and dosage can by those skilled in the art according to the type of the cell and amount be suitable for determine.
In the step C, the chromatin being immobilized obtained in the step B is digested, is fixed
The chromatin fragments of change.Deoxyribonuclease can be used to carry out in the digestion.It is excellent as the deoxyribonuclease
Select I type restriction enzyme, II type restriction enzyme or III type restriction enzyme.The Hi- documented by non-patent literature 2
In C method, because also the restriction enzyme of 4 base sequences of identification to be used to carry out DNA fragmentation in subsequent step, only
The restriction enzyme of 6 base sequences of identification can be used to digest chromatin.The resolution ratio of this digestion method is low, retains
Information content it is small.On the other hand, in the method for the invention, because in subsequent step method, transposase can be interrupted using ultrasound
The methods of method, hydraulic shear cutting method carry out DNA fragmentation, so resolution ratio 4 bases of higher identification can be used in step C
The restriction enzyme of sequence or other nucleases digest chromatin, this make theoretically resolution ratio improve ten number
Times, obtainable chromatin interaction information content is significantly increased.Certainly, 6 base sequences of identification be can also use in step C
Restriction enzyme carries out chromatin digestion.
In the step D, the chromatin fragments being immobilized obtained in the step C are directly connected again
It connects, the chromatin fragments being immobilized reconnected.Herein, it " is directly reconnected " and is referred to: not to described
The chromatin fragments being immobilized carry out biotin labeling, and these segments are reconnected.In addition, in the step C
When the middle progress chromatin digestion using cohesive end restriction enzyme, the obtained chromatin fragments being immobilized have viscosity
End, it is preferable that these cohesive ends repair making unlike the prior art in method of the invention and put down end
End, but cohesive end connection type is used to reconnect by these chromatin fragments, such efficiency is higher than flat end
Connection.Certainly, this method can also using repair after carry out blunt end cloning (but without tie point label as be added band biology
End is glued in the nucleotide reparation of element label) or using flat terminal restriction endonuclease digestion and then carry out blunt end cloning.End
End connection can be carried out for example by using active DNA ligase is connected with end, described to have end connection activity
DNA ligase such as T4DNA ligase, T3DNA ligase, e. coli dna ligase, heat-stable DNA ligase etc..Even
It is reversed answer used in enzyme and substrate amount and reaction condition can by those skilled in the art optionally be suitable for selection.For example,
It can usually be carried out about 1 minute~200 hours in 0.1~10 × ligase buffer solution in 0~80 DEG C (preferably 10~40 DEG C)
(preferably 1~30 hour).
In the step E, release the chromatin fragments of reconnect obtained in the step D being immobilized solid
Fixedization, released dna segment.In the present specification, it " releases immobilization " to refer to: the chromatin fragments being immobilized described in releasing
In, part close to each other on three-dimensional space be fixed state.For example, being by will be on chromatin in the immobilization
Protein cross and in the case where realizing, " the releasing immobilization " refers to that protein goes to be crosslinked.The method that protein goes crosslinking
It is known to the skilled in the art, the method and/or high-temperature process of solution crosslinking can be usually carried out using biology, chemical treatment
The method for solving crosslinking, thus released dna segment.For example, the method as the crosslinking of high-temperature process solution, it can be by by above-mentioned connection
System after reaction is placed in 50~100 DEG C (preferably 60~80 DEG C), 1 minute~200 hours (preferably 1~30 hour) Lai Jinhang eggs
The crosslinking of white matter solution.It, can be by the way that endopeptidase, silk ammonia be added into the system as biology, the method for chemical treatment solution crosslinking
Pepsin, thiol protease, metalloproteinases, aspartic protease, pepsin, trypsase, cathepsin, wood
Melon protease, subtilopeptidase A, Proteinase K, DTT, NaCl, KCl or their combination carry out.It is of course also possible to logical
It crosses and biology is applied in combination, be chemically treated the method for method and the crosslinking of high-temperature process solution that solution is crosslinked to carry out protein solution crosslinking.
In addition, there are in the case where high temperature processing step in the DNA fragmentation amplification step of following step F, the step E can also be with
Step F is carried out together.
In step F, the DNA fragmentation discharged in the step E is expanded, amplified production is obtained.For amplification side
Method is not particularly limited, as long as can obtain for constructing sequencing DNA library as sufficient quantity (such as 0.001~1000ng)
Amplified production.For example, can be suitable few using MDA, MALBAC, NEB-WGA, GenomePlex (preferably MALBAC) etc.
Cell, unicellular or minim DNA amplification method are measured, the actual conditions of these amplification methods can be regarded by those skilled in the art and be needed
It is suitable for selecting.Above-mentioned amplification method is generally based on PCR reaction (polymerase chain reaction) Lai Jinhang, and the PCR reaction is general
Pass through certain PCR response procedures (temperature cycles) Lai Shixian.The PCR response procedures generally comprise denaturation, annealing, extension etc.
Step.The design of primer used in PCR reaction is well known to those skilled in the art, such as can be according to " molecular cloning is real
Test guide " introduction of (J. Pehanorm Brooker (Sambrook.J.) etc. writes, and Huang Peitang etc. translates, the 3rd edition, 2005) is designed,
Or it is designed using computer software (such as Primer Premier6.0 of Premier company exploitation).
In the step G, by amplified production fragmentation obtained in the step F, smaller DNA fragmentation is obtained.In
In this specification, " smaller DNA fragmentation " refer to its size be suitable for building sequencing (such as two generations sequencing, three generations sequencing or four generations
Sequencing) use DNA library, such as Illumina DNA sequencing library.The specific size of " smaller DNA fragmentation " can be such as 10
~50000bp, preferably 50~1000bp.In the Hi-C method documented by non-patent literature 2, it is only suitable for using identifying 4 bases
The restriction enzyme of sequence carries out amplified production fragmentation in the method for the invention, can also still be interrupted using ultrasound
Method, swivel base enzyme process, hydraulic shear cutting method are by the amplified production fragmentation.This improves resolution ratio, can obtain more information.
The technology of amplified production fragmentation is known to the skilled in the art using the above method, can optionally select suitable condition
Come carry out.
In the step H, using smaller DNA fragmentation obtained in the step G as DNA fragmentation to be sequenced, building
DNA library is used in sequencing.Such as standard Illumina DNA small fragment banking process, PCR free method, one-step method can be used
DNA small fragment banking process is waited to construct the sequencing DNA library.The method of various building sequencing DNA libraries is ability
Known to field technique personnel, it can be carried out by those skilled in the art according to routine operation.For example, standard Illumina DNA
Small fragment banking process generally include end repair, end add A, Adapter connection, amplification, amplified production purifying and etc., can
It is carried out in the method recommended according to Illumina company.
It should be noted that a part of product can optionally be taken to carry out next step in above-mentioned steps C~G.
Be sequenced by all or part to the sequencing DNA library constructed using method of the invention, then with dye
Chromaticness DNA primary sequence information is compared, and can be obtained the information for the Chromatin domains that may spatially interact.
Embodiment
With reference to embodiments, the present invention will be described in further detail.It should be appreciated that specific reality described herein
Applying example is to be used to explain the present invention, not limitation of the invention.
Embodiment 1
1. sample process
1.1 crosslinking
1.1.1 1-10 human leukocytes sample is placed in the PBS of 10 μ L and cell suspending liquid drop is made；
1.1.2 the formalin that 10 μ L 4% are added is crosslinked, and 5min is stored at room temperature；
1.1.3 the glycine solution that 5 μ L concentration are 0.25M is added into reaction drop and terminates crosslinking.It stands at room temperature
5min, then ice bath 15min；
1.2 unicellular (a small amount of cell) pickings
Individual cells are chosen using capillary tube method or a small amount of cell is placed in lysate.
Crack formula of liquid:
10mM Tris-HCl pH 7.4
30mM NaCl
0.2%NP-40
10%protease inhibitor cocktail
Sterile water
2. cell cracking
2.1 by the micro- centrifugation of the cell lysis buffer solution containing unicellular sample by liquid pools in tube bottom, ice bath later
45min；
The 2.2 SDS solution for being added 4% make SDS final concentration of 0.35%, 37 DEG C of warm bath 60min；
The 2.3 Triton X-100 solution for being added 20% make Triton X-100 final concentration of 3%, 37 DEG C of warm bath 60min.
3. chromatin digests
3.1 pairs of each samples, be added 8U (international unit) Mbo I and it is final concentration of 1 × NEBuffer
3.1 buffer.37 DEG C warm bath 16 hours.
3.2 inactivation restriction endonucleases.65 DEG C, warm bath 20min.
4.DNA reconnection and DNA, the crosslinking of albumen solution
4.1 pairs of each samples, be added 8U (international unit) T4DNA ligase and final concentration of 1 × connection enzyme buffer
Liquid.16 DEG C warm bath 16 hours.
4.2 65 DEG C warm bath 16 hours.
5.DNA amplification
Using the hundred million health single cell whole genome amplification kits based on MALBAC method, according to kit specification, right
The sample being previously obtained carries out DNA cloning, obtains the amplified production of reconnection DNA.
6.DNA fragmentation
Ultrasound is carried out to amplified production using Diagenode Bioruptor UCD-600 (NGS) to interrupt, and interrupts program
Are as follows: 30 seconds ultrasounds are rested for 30 seconds, 22 circulations.The DNA fragmentation chemical conversion clip size that front is expanded is between 100-700bp
DNA fragmentation.
7. small fragment library construction
It repairs 7.1 ends
It is walked upwards according to following table and reparation reaction system in end is added in product:
Sample is placed in 20e warm bath 30min in Thermomixer.Beckman Agencourt is used after reaction
DNA in AMPure XP Nucleic acid purification kits recovery purifying reaction system, is dissolved in the water of 32 μ L.
7.2 ends add " A "
It is walked to be added in product upwards according to following table and adds " A " reaction system:
Sample is placed in 37 DEG C of warm bath 30min in Thermomixer.Use Beckman Agencourt AMPure XP
DNA in Nucleic acid purification kits recovery purifying reaction system is dissolved in the water of 18 μ L.
7.3 " Adapter " connection:
Walk addition " Adapter " reaction system in product upwards according to following table:
Sample is placed in 20 DEG C of warm bath 15min in Thermomixer.Use Beckman Agencourt AMPure XP
DNA in Nucleic acid purification kits recovery purifying reaction system is dissolved in the water of 30 μ L.
7.4 amplified library
It is walked in product upwards according to following table and following reaction system is added:
The program setting of PCR reaction is as follows:
It is purified using Beckman Agencourt AMPure XP Nucleic acid purification kits；The dissolution of 15 μ L water；Purifying
After measure DNA concentration.
7.5 library quality inspections, upper machine carry out both-end sequencing, obtain data
Different from non-patent literature 1 and 2, the present invention takes reconnection site DNA fragmentation without using biotin fishing, but utilizes letter
Breath credit analysis filters out segment locating for non-reconnection site.Due to having to this in the Hi-C sequencing data analysis method of standard
Segment filtering (filter condition be in non-patent literature 1 and 2 in order to remove in library it is remaining by biotin labeling not
Reconnection segment), so the library that the present invention generates can carry out bioinformatic analysis and obtain without additional filter condition
Interaction information of the chromosome on three-dimensional space.
7.6 obtain information content and in existing literature slender karyon Hi-C compare
The present invention carries out chromatin using Mbo I (recognition site be GATC but to Dam, Dcm and CpG methyl-sensitive) and disappears
Change, under the sequencing amount that 14M-19M both-end is sequenced reads pairs, capturing the interaction logarithm between Chromatin domains is 270-
331K.Compare in contrast, in the slender karyon Hi-C of non-patent literature 2 using Dpn II (recognition site be also GATC but to Dam,
Dcm methyl-sensitive, that is, its recognition site mathematics should be higher than that Mbo I, therefore should theoretically obtain more digestion pieces
Section and Chromatin domains interaction information) chromatin digestion is carried out, it can only be captured under the sequencing amount that 13M both-end is sequenced reads pairs
To the interaction number between the Chromatin domains of 12K；The slender karyon of remaining in non-patent literature 2 using II restriction endonuclease of Bgl into
The digestion of row chromatin, the phase interaction capturing Chromatin domains under the sequencing amount that 5.5M-15.3M both-end is sequenced reads betweens
It is 11.7k-30.6k with number.The slender of non-patent literature 2 is significantly higher than using the chromatin conformation information of acquisition of the invention
The method of karyon Hi-C.
It should also be noted that, it is implementable and it is unobvious violate purport of the invention under the premise of, in the present specification
The combination of any technical characteristic or technical characteristic described in composition part as a certain technical solution equally can also be applied
In other technical solutions；Also, it is implementable and it is unobvious violate purport of the invention under the premise of, as different technologies scheme
Composition part described in can also be combined in any way between technical characteristic, to constitute other technical solutions.This
Invention is also contained under above situation the technical solution as obtained from combination, and these technical solutions are equivalent to and are documented in this
In specification.
The preferred embodiment of the present invention has shown and described in above description, as previously described, it should be understood that the present invention is not office
Be limited to form disclosed herein, should not be regarded as an exclusion of other examples, and can be used for various other combinations, modification and
Environment, and can be changed within that scope of the inventive concept describe herein by the above teachings or related fields of technology or knowledge
It is dynamic.And the modifications and changes that those skilled in the art are carried out do not depart from the spirit and scope of the present invention, then it all should be in the present invention
In the protection scope of appended claims.
Industrial applicibility
According to the present invention it is possible to carry out the detection of chromatin conformation to the sample of a small amount of cell.Cell type includes but not
It is limited to: zooblast, plant cell, microbial cell, virus, cancer cell；These cell origins include but is not limited to: primary training
It supports, cell line culture, tissue, organism, environmental sources, fossil.
According to the present invention it is possible between more different cells chromosomal conformation difference.Difference packet between these cells
Include but be not limited to: different plant species source, Different Organs source, different cell category, the different cell cycles, different developmental phases,
Different condition of culture, different disposal condition, different cell individuals.
According to the present invention it is possible to analyze between the variation of endochrome conformation change and other DNA, RNA, protein
Connection.These variation include but is not limited to following aspect: DNA mutation, DNA methylation variation, gene knockout, gene knock-in,
Transgenosis, rna expression variation, RNA silencing, microRNA expression variation, long non-coding RNA expression variation, 16s rDNA variation,
MRNA expression variation, rRNA expression variation, the variation of RNA conformation change, DNA conformation change, DNA controlling element, chromosome
Exception, chromosome deficiency, chromosome repetition, chromosome translocation, chromosomal conformation variation, CNV, protein expression variation, antigen are anti-
Body variation, secretory protein variation, memebrane protein variation.
According to the present invention it is possible to cyto-chromatin conformation and specific gene, DNA, RNA, the relationship between albumen are studied, point
Analyse the interaction between intracellular specific protein and cell nuclear dna, RNA.
According to the present invention it is possible to which research cell characteristics or function is used in combination with the method for other researching DNAs, RNA, albumen
The function of energy and chromatin conformation, DNA, RNA and protein.These other researching DNAs, RNA, albumen method include but
Be not limited to: genetic chip, QPCR, generation sequencing, the sequencing of two generations, three generations's sequencing, the sequencing of four generations, gene sequencing, gene order-checking,
Macro gene order-checking, exon sequencing, introne sequencing, target gene capture sequencing, RNA sequencing, express spectra sequencing, transcript profile
Sequencing, the sequencing of tiny RNA transcript profile, microRNA, the sequencing of macro transcript profile, LncRNA sequencing, oncogene sequencing, Oncogenome are surveyed
Sequence, Bisulfite methylation sequencing, ChIP-DNA sequencing, MeDIP sequencing, RRBS sequencing, Target-BS sequencing, hmC sequencing.
Claims (15)
1. a kind of method for constructing the library Hi-C, this method include the following steps:
Step B: a small amount of chromatin being immobilized is obtained, a small amount of chromatin being immobilized is 10-6~102Ng's
Chromatin, in terms of naked DNA；
Step C: the chromatin being immobilized obtained in the step B is digested, the chromatin piece being immobilized
Section；
Step D: the chromatin fragments being immobilized obtained in the step C are directly reconnected, are connected again
The chromatin fragments being immobilized connect；
Step E: the chromatin fragments of reconnect obtained in the step D being immobilized are made to release immobilization, released dna
Segment；
Step F: the DNA fragmentation discharged in the step E is expanded, amplified production is obtained；And
Step H: using the amplified production as DNA fragmentation to be sequenced, sequencing DNA library is constructed.
2. according to the method described in claim 1, wherein, a small amount of chromatin being immobilized is 10-5The dye of~10ng
Chromaticness, in terms of naked DNA.
3. according to the method described in claim 1, wherein, being fixed using deoxyribonuclease to described in the step C
The chromatin of change is digested.
4. according to the method described in claim 3, wherein, the deoxyribonuclease is I type restriction enzyme, II type limit
Property restriction endonuclease processed or III type restriction enzyme.
5. according to the method described in claim 1, wherein, in the step D using viscous end or blunt end cloning method by institute
The chromatin fragments being immobilized obtained in step C are stated to be reconnected.
6. according to the method described in claim 1, its further include:
Step G: by amplified production fragmentation obtained in the step F, smaller DNA fragmentation is obtained；And
In the step H, using smaller DNA fragmentation obtained in the step G as DNA fragmentation to be sequenced, building sequencing is used
DNA library.
7. according to the method described in claim 6, wherein, interrupting method, swivel base enzyme process, restriction endonuclease using ultrasound in the step G
Enzyme cutting method or hydraulic shear cutting method are by the amplified production fragmentation.
8. according to the method described in claim 7, wherein, the size of smaller DNA fragmentation obtained in the step G is 50~
1000bp。
9. according to the method described in claim 1, its further include:
Step A: obtaining the cell that a small amount of chromatin is immobilized, and the cell that a small amount of chromatin is immobilized is 1~
10000 cells；And
In the step B, the cell obtained in the step A is cracked, obtains a small amount of chromatin being immobilized.
10. according to the method described in claim 9, wherein, the cell that a small amount of chromatin is immobilized is 1~1000
Cell.
11. according to the method described in claim 9, wherein, the cell that a small amount of chromatin is immobilized is individual cells.
12. according to the method described in claim 9, wherein, the step A includes:
Step A-1: immobilizing the chromatin of a certain amount of cell, obtain a certain amount of chromatin be immobilized it is thin
Born of the same parents；And
Step A-2: a small amount of chromatin of picking in the cell that a certain amount of chromatin obtained from the step A-1 is immobilized
The cell being immobilized；
It is described it is a certain amount of be 105More than a.
13. according to the method for claim 12, wherein it is described it is a certain amount of be 106~108It is a.
14. according to the method described in claim 9, wherein, the step A includes:
Step A-3: immobilizing the chromatin of a small amount of cell, obtains the cell that a small amount of chromatin is immobilized, institute
Stating a small amount of cell is 1~10000 cell.
15. a kind of method for measuring the Chromatin domains spatially to interact, this method comprises:
The library Hi-C is constructed using method described in any one of claim 1~14；And
The all or part in the library Hi-C is sequenced, and by obtained information and chromatin dna primary sequence information
It is compared.
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