CN105441548A - Zebrafish embryo single-cell hypersensitive-site high-throughput sequencing experiment method - Google Patents
Zebrafish embryo single-cell hypersensitive-site high-throughput sequencing experiment method Download PDFInfo
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Abstract
The invention relates to a zebrafish embryo single-cell hypersensitive-site high-throughput sequencing experiment method. The method comprises firstly pretreating a zebrafish embryo sample, then transferring the embryo to a tube containing a lysis buffer, and performing lysis on the embryo by using a wide-mouth gun head; and then performing deoxyribonuclease I enzyme digestion reaction, extracting and purifying DNA, constructing a library by utilizing a library-establishing kit, and then selecting the demanded size of the library for high-throughput sequencing and bioinformation analysis through agarose gel electrophoretic separation. Compared with the prior art, the method employs a process of adding circular vector DNA during precipitation for enriching DNA, and thus DNase I hypersensitive-site high-throughput sequencing at a single-cell level is performed. By utilizing the method, the transcription regulation-control area difference among cells can be observed, cell heteroplasmy is analyzed, and gene transcription regulation and control change and mechanism research during embryo development are relatively well observed.
Description
Technical field
The invention belongs to biology field, especially relate to DNaseI hypersensitive site and the research of gene transcription regulation related mechanism on full-length genome in zebrafish embryo early development process.
Background technology
Gene regulating is one of central topic of modern molecular biology research.Because understand growth and development of plants and animals rule, Morphologic Characteristics and biological function, just must make the Time and place concept of expression regulation clear, grasp mechanisms of gene regulation, just equals to have grasped one the key disclosing biology secret.And the expression of gene is by multiple function transcriptional elements (transcriptionelements, TEs) combination of binding site regulates and controls, these transcriptional regulatory elements are combined in (transcriptionfactorbindingsites on the special site in gene promoter sequence, TFBSs), the vital role of regulatory transcription is played.The experiment of DNaseI hypersensitive site can provide important transcriptional regulatory element calmodulin binding domain CaM and chromatin Structure change.Traditional DNase-seq is mainly limited to the full-length genome DNaseI hypersensitive site information obtaining a large amount of cell, and singlecellDNase-seq (scDNase-seq) is a kind of method detecting the DNaseI hypersensitive site of individual cells or a small amount of cell.In fact, even originate identical individual cells, due to random bioprocess and environmental perturbation, also there are differences in many aspects each other, i.e. the heterogeneity of cell.ScDNase-seq contributes to us and dissects cell heterogeneity, the difference providing the transcriptional regulatory element between cell with cell to be combined, and is applicable to the gene expression regulation Mechanism Study of embryo's early differentiation and growth.
Zebra fish is the emerging model animal of research developmental biology.The plurality of advantages such as zebra fish is raised easily owing to having, and embryo is transparent, in vitro fertilization, and genetic tool is ripe, have become the emerging model animal of research vertebrate animal development and human inheritance's disease in recent years.Research finds, zebra fish have shared the protein coding gene of the mankind 70%, and has 84% can have found corresponding gene in zebra fish in study of human disease-related gene position, and also compares shortcoming at the high-throughout molecular biology sequencing technologies of zebra fish system at present.
Current unicellular DNase-seq is just in cell sample, and the research for transcriptional control original paper and gene expression regulation is also only limited to cell levels, does not also relate to the research of zebra fish body early embryo.
Summary of the invention
The present invention is that the one that the heterogeneity in order to reduce cell is set up is applicable to zebrafish embryo unicellular chromatin hypersensitive site high-flux sequence (SinglecellDNase-seq) experimental technique.
Object of the present invention can be achieved through the following technical solutions:
Zebrafish embryo unicellular chromatin hypersensitive site high-flux sequence (SinglecellDNase-seq) experimental technique, the method comprises the following steps:
(1) zebrafish embryo sample experiment pre-treatment:
The embryo getting a zebra fish cell stage adds the PBS detergent washing containing protein inhibitor 3 times of precooling, in PBS washing composition, the content of protein inhibitor cocktail is 1X, and described protein inhibitor cocktail is made up of AEBSF, EDTA, Leupeptin and PepstatinA;
(2) lysing cell gets nucleus:
Zebrafish embryo one cell stage sample through pre-treatment proceeds to the lysis buffer (10mMTris-Hcl that 32ul precooling is housed, pH7.5,10mMNaCl, 3mMMgCl2, in the PCR pipe of 0.1ml 0.1%TritonX-100), repeatedly blow and beat embryo with the 100ul pipettor gun head of wide-mouth on ice, until the cracking of embryo's inner cell, avoid bubble and foam to produce, continue cracking 30min on ice;
(3) deoxyribonuclease DNaseI endonuclease reaction:
The DNaseI of the 0.033U/ul that 8ul has diluted is added in the nucleus liquid of above-mentioned cracking, 37 degree of water-bath 5min after vortex 2s gently, add the termination reaction liquid (10mMTris-Hcl that 80ul contains Proteinase K immediately, pH7.5,10mMNaCl, 0.15%SDS, 10mMEDTA), of short duration centrifugal after mixing, add 6ul circular vectors DNA (pUC19), 55 degree of water-bath 1h.DNaseI comes from 5ulDNaseI mother liquor (10U/ul) and adds lysis buffer described in 145ul step (2).Proteinase K concentration is 20mg/ml.Circular vectors DNA (pUC19) concentration is 5ng/ul;
(4) purification enzyme cuts DNA:
Above-mentioned gained liquid proceeds in the PhaseLockGel pipe of 1.5ml, add 125ul extracting to tuck in (mixed solution of the phenol of volume ratio 25:24:1, chloroform and primary isoamyl alcohol), concuss 1min, 14000rpm, the centrifugal 5min of normal temperature, get 125ul supernatant in the centrifuge tube of new 1.5ml, add 1ul20mg/ml glycogen, 12.5ul3MNaOAcpH5.2,340ml dehydrated alcohol,-80 degree precipitation 15min, 13000rpm, 4 degree of centrifugal 15min abandon supernatant 70% ethanol and wash once, 13000rpm, 4 degree of centrifugal 5min remove supernatant, add 10ulQiagen elutriant after drying;
(5) library construction and order-checking:
Carry out quantitatively with Qubit2.0 to gained sample, then library is built according to the storehouse test kit specification sheets of building of RubiconGenomics company, gained library is electrophoretic separation in 2% agarose gel, 120V, 30min, choose after glue is cut in 150-400bp position and carry out library recovery with Qiagen recovery test kit, library will be built and carry out bioanalyzer analysis, determine Library Quality, the 125PE of Hiseq2500 carries out high-flux sequence, and result carries out analysis of biological information.
Compared with prior art, the present invention has the following advantages and beneficial effect:
The present invention carrys out enrichment DNA amount mainly through adding circular vectors DNA in precipitation process, thus the DNaseI hypersensitive site high throughput sequencing technologies that can carry out in individual cells level, this method can observe the difference of the transcription regulating region between cell and cell, dissect the heterogeneity of cell, change and the Mechanism Study of gene transcription regulation in embryo development procedure can better be observed.
Embodiment
The present invention relates to zebrafish embryo unicellular chromatin hypersensitive site high-flux sequence experimental technique, first carry out zebrafish embryo Sample pretreatment, then embryo is gone in the pipe containing lysis buffer, with wide-mouth rifle head cracking embryo in ice bath; Carry out deoxyribonuclease I endonuclease reaction again, extracting and purifying DNA, utilize to build after storehouse test kit builds library and select required library size to carry out high-flux sequence and analysis of biological information by agarose gel electrophoresis separation.
The present invention carrys out enrichment DNA amount mainly through adding circular vectors DNA in precipitation process, thus the DNaseI hypersensitive site high throughput sequencing technologies that can carry out in individual cells level, this method can observe the difference of the transcription regulating region between cell and cell, dissect the heterogeneity of cell, change and the Mechanism Study of gene transcription regulation in embryo development procedure can better be observed.
Below in conjunction with specific embodiment, technical solution of the present invention is described in detail.
Embodiment 1
Get the zebrafish embryo one piece of the one cell stage of firm after fertilization.Concrete grammar is as follows:
(1) zebrafish embryo sample experiment pre-treatment:
The embryo getting a zebra fish cell stage adds the PBS detergent washing containing protein inhibitor 3 times of precooling, in PBS washing composition, the content of protein inhibitor cocktail is 1X, and described protein inhibitor cocktail is made up of AEBSF, EDTA, Leupeptin and PepstatinA;
(2) lysing cell gets nucleus:
Zebrafish embryo one cell stage sample through pre-treatment proceeds to the lysis buffer (10mMTris-Hcl that 32ul precooling is housed, pH7.5,10mMNaCl, 3mMMgCl2, in the PCR pipe of 0.1ml 0.1%TritonX-100), repeatedly blow and beat embryo with the 100ul pipettor gun head of wide-mouth on ice, until the cracking of embryo's inner cell, avoid bubble and foam to produce, continue cracking 30min on ice;
(3) deoxyribonuclease DNaseI endonuclease reaction:
The DNaseI of the 0.033U/ul that 8ul has diluted is added in the nucleus liquid of above-mentioned cracking, 37 degree of water-bath 5min after vortex 2s gently, add the termination reaction liquid (10mMTris-Hcl that 80ul contains 20mg/ml Proteinase K 1ul immediately, pH7.5,10mMNaCl, 0.15%SDS, 10mMEDTA), of short duration centrifugal after mixing, add 6ul5ng/ul circular vectors DNA (pUC19), 55 degree of water-bath 1h.DNaseI comes from 5ulDNaseI mother liquor (10U/ul) and adds lysis buffer described in 145ul step (2);
(4) purification enzyme cuts DNA:
Above-mentioned gained liquid proceeds in the PhaseLockGel pipe of 1.5ml, add 125ul extracting to tuck in (mixed solution of the phenol of volume ratio 25:24:1, chloroform and primary isoamyl alcohol), concuss 1min, 14000rpm, the centrifugal 5min of normal temperature, get 125ul supernatant in the centrifuge tube of new 1.5ml, add 1ul20mg/ml glycogen, 12.5ul3MNaOAcpH5.2,340ml dehydrated alcohol,-80 degree precipitation 15min, 13000rpm, 4 degree of centrifugal 15min abandon supernatant 70% ethanol and wash once, 13000rpm, 4 degree of centrifugal 5min remove supernatant, add 10ulQiagen elutriant after drying;
(5) library construction and order-checking:
Carry out quantitatively with Qubit2.0 to gained sample, then library is built according to the storehouse test kit specification sheets of building of RubiconGenomics company, gained library is electrophoretic separation in 2% agarose gel, 120V, 30min, choose after glue is cut in 150-400bp position and carry out library recovery with Qiagen recovery test kit, library will be built and carry out bioanalyzer analysis, determine Library Quality, the 125PE of Hiseq2500 carries out high-flux sequence, and result carries out analysis of biological information.
Embodiment 2
This method is applied to a small amount of cell of zebra fish body early embryo, gets 256 cell stages of successful fertilization, 1k cell stage and each one piece of Dome/30%epiboly cell stage zebrafish embryo, comprise the following steps:
(1) zebrafish embryo sample experiment pre-treatment:
Get each one piece of zebra fish embryo in each period, add the PBS detergent washing 3 times containing protein inhibitor of precooling, in PBS washing composition, the content of protein inhibitor cocktail is 1X, and described protein inhibitor cocktail is made up of AEBSF, EDTA, Leupeptin and PepstatinA;
(2) lysing cell gets nucleus:
Zebrafish embryo one cell stage sample through pre-treatment proceeds to the lysis buffer (10mMTris-Hcl that 32ul precooling is housed, pH7.5,10mMNaCl, 3mMMgCl2, in the PCR pipe of 0.1ml 0.1%TritonX-100), repeatedly blow and beat embryo with the 100ul pipettor gun head of wide-mouth on ice, until the cracking of embryo's inner cell, avoid bubble and foam to produce, continue cracking 30min on ice;
(3) deoxyribonuclease DNaseI endonuclease reaction:
The DNaseI of the 0.033U/ul that 8ul has diluted is added in the nucleus liquid of above-mentioned cracking, 37 degree of water-bath 5min after vortex 2s gently, add the termination reaction liquid (10mMTris-Hcl that 80ul contains 20mg/ml Proteinase K 1ul immediately, pH7.5,10mMNaCl, 0.15%SDS, 10mMEDTA), I comes from 5ulDNaseI mother liquor (10U/ul) and adds lysis buffer described in 145ul step (2);
(4) purification enzyme cuts DNA:
Above-mentioned gained liquid proceeds in the PhaseLockGel pipe of 1.5ml, add 125ul extracting to tuck in (mixed solution of the phenol of volume ratio 25:24:1, chloroform and primary isoamyl alcohol), concuss 1min, 14000rpm, the centrifugal 5min of normal temperature, get 125ul supernatant in the centrifuge tube of new 1.5ml, add 1ul20mg/ml glycogen, 12.5ul3MNaOAcpH5.3,340ml dehydrated alcohol,-80 degree precipitation 15min, 13000rpm, 4 degree of centrifugal 15min abandon supernatant 70% ethanol and wash once, 13000rpm, 4 degree of centrifugal 5min remove supernatant, add 10ulQiagen elutriant after drying;
(5) library construction and order-checking:
Carry out quantitatively with Qubit2.0 to gained sample, then library is built according to the storehouse test kit specification sheets of building of RubiconGenomics company, gained library is electrophoretic separation in 2% agarose gel, 120V, 30min, choose after glue is cut in 150-400bp position and carry out library recovery with Qiagen recovery test kit, library will be built and carry out bioanalyzer analysis, determine Library Quality, the 125PE of Hiseq2500 carries out high-flux sequence, and result carries out analysis of biological information.
Above-mentioned is can understand and use invention for ease of those skilled in the art to the description of embodiment.Person skilled in the art obviously easily can make various amendment to these embodiments, and General Principle described herein is applied in other embodiments and need not through performing creative labour.Therefore, the invention is not restricted to above-described embodiment, those skilled in the art, according to announcement of the present invention, do not depart from improvement that scope makes and amendment all should within protection scope of the present invention.
Claims (7)
1. zebrafish embryo unicellular chromatin hypersensitive site high-flux sequence (SinglecellDNase-seq) experimental technique, it is characterized in that, the method comprises the following steps:
(1) zebrafish embryo sample experiment pre-treatment:
The embryo getting a zebra fish cell stage adds the PBS detergent washing containing protein inhibitor 3 times of precooling;
(2) lysing cell gets nucleus:
Zebrafish embryo one cell stage sample through pre-treatment proceeds in the PCR pipe of the 0.1ml of the lysis buffer that 32ul precooling is housed, repeatedly embryo is blown and beaten on ice with the 100ul pipettor gun head of wide-mouth, until the cracking of embryo's inner cell, avoid bubble and foam to produce, continue cracking 30min on ice;
(3) deoxyribonuclease DNaseI endonuclease reaction:
The DNaseI of the 0.033U/ul that 8ul has diluted is added in the nucleus liquid of above-mentioned cracking, 37 degree of water-bath 5min after vortex 2s gently, add the termination reaction liquid that 80ul contains Proteinase K immediately, of short duration centrifugal after mixing, add 6ul circular vectors DNA (pUC19), 55 degree of water-bath 1h;
(4) purification enzyme cuts DNA:
Above-mentioned gained liquid proceeds in the PhaseLockGel pipe of 1.5ml, add 125ul extracting to tuck in, concuss 1min, maximum velocity centrifugation 5min, get 125ul supernatant in the centrifuge tube of new 1.5ml, add 1ul glycogen, 12.5ulNaOAc, 340ml dehydrated alcohol,-80 degree precipitation 15min, centrifugal 15min abandons supernatant 70% ethanol and washes once, and centrifugal 5min removes supernatant, adds 10ulQiagen elutriant after drying;
(5) library construction and order-checking:
Carry out quantitatively with Qubit2.0 to gained sample, then library is built according to the storehouse test kit specification sheets of building of RubiconGenomics company, gained library is electrophoretic separation in 2% agarose gel, choose after glue is cut in 150-400bp position and carry out library recovery with Qiagen recovery test kit, library will be built and carry out bioanalyzer analysis, determine Library Quality, the 125PE of Hiseq2500 carries out high-flux sequence, and result carries out analysis of biological information.
2. zebrafish embryo according to claim 1 unicellular chromatin hypersensitive site high-flux sequence experimental technique, it is characterized in that, in step (1), in described PBS washing composition, the content of protein inhibitor cocktail is 1X, and described protein inhibitor cocktail is made up of AEBSF, EDTA, Leupeptin and PepstatinA.
3. zebrafish embryo according to claim 1 unicellular chromatin hypersensitive site high-flux sequence experimental technique, is characterized in that, the lysis buffer described in step (2) specifically comprises: 10mMTris-Hcl, pH7.5,10mMNaCl, 3mMMgCl2,0.1%TritonX-100.
4. zebrafish embryo according to claim 1 unicellular chromatin hypersensitive site high-flux sequence experimental technique, it is characterized in that, the DNaseI described in step (3) comes from 5ulDNaseI mother liquor (10U/ul) and adds lysis buffer described in 145ul step (2); Termination reaction liquid specifically comprises: 10mMTris-Hcl, pH7.5,10mMNaCl, 0.15%SDS, 10mMEDTA; Proteinase K concentration is 20mg/ml; Circular vectors DNA (pUC19) concentration is 5ng/ul.
5. zebrafish embryo according to claim 1 unicellular chromatin hypersensitive site high-flux sequence experimental technique, it is characterized in that, the mixed solution into the phenol of volume ratio 25:24:1, chloroform and primary isoamyl alcohol is tucked in extracting described in step (4), and centrifugal rotational speed is 14000rpm, normal temperature.
6. zebrafish embryo according to claim 1 unicellular chromatin hypersensitive site high-flux sequence experimental technique, is characterized in that, step (4) glycogen concentration is 20mg/ml.NaOAc is 3MpH5.2; Centrifugal rotational speed is 13000rpm, 4 degree.
7. zebrafish embryo according to claim 1 unicellular chromatin hypersensitive site high-flux sequence (SinglecellDNase-seq) experimental technique, is characterized in that, the electrophoresis described in step (5) is 120V, 30min.
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Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652365A (en) * | 2019-02-01 | 2019-04-19 | 南京大学 | A kind of preparation method of zebrafish embryo single cell suspension |
| CN113005145A (en) * | 2021-03-09 | 2021-06-22 | 同济大学 | Specific antibody-independent method for capturing binding sites of TF on whole genome |
| CN117417882A (en) * | 2023-10-18 | 2024-01-19 | 中国科学院水生生物研究所 | Preparation method and equipment of single cell suspension of zebra fish embryo |
| WO2024139531A1 (en) * | 2022-12-26 | 2024-07-04 | 武汉希望组生物科技有限公司 | Kit and method for extracting dna from blood or cell sample |
-
2015
- 2015-12-21 CN CN201510970471.XA patent/CN105441548A/en active Pending
Non-Patent Citations (4)
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| WEI-PING ZENG: "Rapid and Unambiguous Detection of DNase I Hypersensitive Site in Rare Population of Cells", 《PLOS ONE》 * |
| WENFEI JIN等: "Genome-wide detection of DNase 1 hypersensitive sites in single cells and FFPE tissue sample", 《NATURE》 * |
| 于文琪: "染色质DNaseⅠ超敏感位点的定位及其转录调控功能的研究进展", 《医学研究生学报》 * |
| 刘超英等: "《中枢神经系统疾病蛋白质组学》", 31 December 2010 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109652365A (en) * | 2019-02-01 | 2019-04-19 | 南京大学 | A kind of preparation method of zebrafish embryo single cell suspension |
| CN113005145A (en) * | 2021-03-09 | 2021-06-22 | 同济大学 | Specific antibody-independent method for capturing binding sites of TF on whole genome |
| WO2024139531A1 (en) * | 2022-12-26 | 2024-07-04 | 武汉希望组生物科技有限公司 | Kit and method for extracting dna from blood or cell sample |
| CN117417882A (en) * | 2023-10-18 | 2024-01-19 | 中国科学院水生生物研究所 | Preparation method and equipment of single cell suspension of zebra fish embryo |
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