CN113215141A - Bacterial HI-C genome and plasmid conformation capture method - Google Patents
Bacterial HI-C genome and plasmid conformation capture method Download PDFInfo
- Publication number
- CN113215141A CN113215141A CN202110204762.3A CN202110204762A CN113215141A CN 113215141 A CN113215141 A CN 113215141A CN 202110204762 A CN202110204762 A CN 202110204762A CN 113215141 A CN113215141 A CN 113215141A
- Authority
- CN
- China
- Prior art keywords
- minutes
- bacterial
- bacteria
- dna
- solution
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
- 230000001580 bacterial effect Effects 0.000 title claims abstract description 55
- 238000000034 method Methods 0.000 title claims abstract description 23
- 239000013612 plasmid Substances 0.000 title claims abstract description 15
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 claims abstract description 28
- 238000004132 cross linking Methods 0.000 claims abstract description 20
- 102000004190 Enzymes Human genes 0.000 claims abstract description 16
- 108090000790 Enzymes Proteins 0.000 claims abstract description 16
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 claims abstract description 16
- 238000006243 chemical reaction Methods 0.000 claims abstract description 16
- 229960002685 biotin Drugs 0.000 claims abstract description 14
- 235000020958 biotin Nutrition 0.000 claims abstract description 14
- 239000011616 biotin Substances 0.000 claims abstract description 14
- 108010077544 Chromatin Proteins 0.000 claims abstract description 13
- 210000003483 chromatin Anatomy 0.000 claims abstract description 13
- 238000011049 filling Methods 0.000 claims abstract description 5
- 241000894006 Bacteria Species 0.000 claims description 56
- 239000000243 solution Substances 0.000 claims description 38
- 239000007788 liquid Substances 0.000 claims description 32
- 108020004414 DNA Proteins 0.000 claims description 31
- 239000000203 mixture Substances 0.000 claims description 30
- 238000012258 culturing Methods 0.000 claims description 26
- 239000006228 supernatant Substances 0.000 claims description 26
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 claims description 19
- 239000002244 precipitate Substances 0.000 claims description 17
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 16
- 229940088598 enzyme Drugs 0.000 claims description 15
- 239000007984 Tris EDTA buffer Substances 0.000 claims description 14
- 239000006166 lysate Substances 0.000 claims description 13
- 239000008188 pellet Substances 0.000 claims description 13
- 238000002156 mixing Methods 0.000 claims description 11
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 10
- 239000008098 formaldehyde solution Substances 0.000 claims description 10
- IJGRMHOSHXDMSA-UHFFFAOYSA-N Atomic nitrogen Chemical compound N#N IJGRMHOSHXDMSA-UHFFFAOYSA-N 0.000 claims description 8
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 claims description 8
- 239000006137 Luria-Bertani broth Substances 0.000 claims description 8
- 239000013049 sediment Substances 0.000 claims description 8
- 102000016943 Muramidase Human genes 0.000 claims description 7
- 108010014251 Muramidase Proteins 0.000 claims description 7
- 108010062010 N-Acetylmuramoyl-L-alanine Amidase Proteins 0.000 claims description 7
- 229960000274 lysozyme Drugs 0.000 claims description 7
- 239000004325 lysozyme Substances 0.000 claims description 7
- 235000010335 lysozyme Nutrition 0.000 claims description 7
- 238000002360 preparation method Methods 0.000 claims description 7
- 238000005406 washing Methods 0.000 claims description 7
- 238000007664 blowing Methods 0.000 claims description 6
- 101100116420 Aedes aegypti DEFC gene Proteins 0.000 claims description 5
- 108020000946 Bacterial DNA Proteins 0.000 claims description 5
- 239000000047 product Substances 0.000 claims description 5
- 239000011541 reaction mixture Substances 0.000 claims description 5
- 108010067770 Endopeptidase K Proteins 0.000 claims description 4
- 239000004471 Glycine Substances 0.000 claims description 4
- 229920002527 Glycogen Polymers 0.000 claims description 4
- 239000006142 Luria-Bertani Agar Substances 0.000 claims description 4
- 102000006382 Ribonucleases Human genes 0.000 claims description 4
- 108010083644 Ribonucleases Proteins 0.000 claims description 4
- VMHLLURERBWHNL-UHFFFAOYSA-M Sodium acetate Chemical compound [Na+].CC([O-])=O VMHLLURERBWHNL-UHFFFAOYSA-M 0.000 claims description 4
- 230000001079 digestive effect Effects 0.000 claims description 4
- 235000011389 fruit/vegetable juice Nutrition 0.000 claims description 4
- 229940096919 glycogen Drugs 0.000 claims description 4
- 229910052757 nitrogen Inorganic materials 0.000 claims description 4
- 239000001632 sodium acetate Substances 0.000 claims description 4
- 235000017281 sodium acetate Nutrition 0.000 claims description 4
- 238000007605 air drying Methods 0.000 claims description 3
- 230000015556 catabolic process Effects 0.000 claims description 3
- 238000005119 centrifugation Methods 0.000 claims description 3
- 238000006731 degradation reaction Methods 0.000 claims description 3
- 238000007598 dipping method Methods 0.000 claims description 3
- 239000012530 fluid Substances 0.000 claims description 3
- 238000002844 melting Methods 0.000 claims description 3
- 230000008018 melting Effects 0.000 claims description 3
- 238000003756 stirring Methods 0.000 claims description 3
- 238000002205 phenol-chloroform extraction Methods 0.000 claims description 2
- 239000012634 fragment Substances 0.000 abstract description 6
- 238000010276 construction Methods 0.000 abstract description 4
- 238000001976 enzyme digestion Methods 0.000 abstract description 4
- YTRQFSDWAXHJCC-UHFFFAOYSA-N chloroform;phenol Chemical compound ClC(Cl)Cl.OC1=CC=CC=C1 YTRQFSDWAXHJCC-UHFFFAOYSA-N 0.000 abstract description 3
- 238000000605 extraction Methods 0.000 abstract description 3
- 238000012165 high-throughput sequencing Methods 0.000 abstract description 3
- 230000003993 interaction Effects 0.000 abstract description 3
- 108090000623 proteins and genes Proteins 0.000 abstract description 3
- 102000004169 proteins and genes Human genes 0.000 abstract description 3
- 238000012163 sequencing technique Methods 0.000 abstract description 3
- 108020004707 nucleic acids Proteins 0.000 abstract description 2
- 102000039446 nucleic acids Human genes 0.000 abstract description 2
- 150000007523 nucleic acids Chemical class 0.000 abstract description 2
- 238000007400 DNA extraction Methods 0.000 abstract 1
- 230000009089 cytolysis Effects 0.000 abstract 1
- 230000029087 digestion Effects 0.000 abstract 1
- 230000014509 gene expression Effects 0.000 abstract 1
- 238000012795 verification Methods 0.000 abstract 1
- 239000011324 bead Substances 0.000 description 18
- 239000000872 buffer Substances 0.000 description 4
- 238000000746 purification Methods 0.000 description 3
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000010009 beating Methods 0.000 description 2
- 210000000349 chromosome Anatomy 0.000 description 2
- 238000005516 engineering process Methods 0.000 description 2
- 230000008014 freezing Effects 0.000 description 2
- 238000007710 freezing Methods 0.000 description 2
- 238000007885 magnetic separation Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- 102000012410 DNA Ligases Human genes 0.000 description 1
- 108010061982 DNA Ligases Proteins 0.000 description 1
- 102000004594 DNA Polymerase I Human genes 0.000 description 1
- 108010017826 DNA Polymerase I Proteins 0.000 description 1
- 108010034791 Heterochromatin Proteins 0.000 description 1
- 102000003960 Ligases Human genes 0.000 description 1
- 108090000364 Ligases Proteins 0.000 description 1
- 241000699670 Mus sp. Species 0.000 description 1
- 229920001213 Polysorbate 20 Polymers 0.000 description 1
- 108010090804 Streptavidin Proteins 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 238000000246 agarose gel electrophoresis Methods 0.000 description 1
- 230000003321 amplification Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 210000004027 cell Anatomy 0.000 description 1
- 239000007795 chemical reaction product Substances 0.000 description 1
- 238000003776 cleavage reaction Methods 0.000 description 1
- 238000005336 cracking Methods 0.000 description 1
- 238000005520 cutting process Methods 0.000 description 1
- RGWHQCVHVJXOKC-SHYZEUOFSA-J dCTP(4-) Chemical compound O=C1N=C(N)C=CN1[C@@H]1O[C@H](COP([O-])(=O)OP([O-])(=O)OP([O-])([O-])=O)[C@@H](O)C1 RGWHQCVHVJXOKC-SHYZEUOFSA-J 0.000 description 1
- HAAZLUGHYHWQIW-KVQBGUIXSA-N dGTP Chemical compound C1=NC=2C(=O)NC(N)=NC=2N1[C@H]1C[C@H](O)[C@@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)O1 HAAZLUGHYHWQIW-KVQBGUIXSA-N 0.000 description 1
- NHVNXKFIZYSCEB-XLPZGREQSA-N dTTP Chemical compound O=C1NC(=O)C(C)=CN1[C@@H]1O[C@H](COP(O)(=O)OP(O)(=O)OP(O)(O)=O)[C@@H](O)C1 NHVNXKFIZYSCEB-XLPZGREQSA-N 0.000 description 1
- 230000007547 defect Effects 0.000 description 1
- 238000001514 detection method Methods 0.000 description 1
- 239000012154 double-distilled water Substances 0.000 description 1
- 230000002708 enhancing effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- 210000004458 heterochromatin Anatomy 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000002054 inoculum Substances 0.000 description 1
- 238000000464 low-speed centrifugation Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000003199 nucleic acid amplification method Methods 0.000 description 1
- 239000002773 nucleotide Substances 0.000 description 1
- 125000003729 nucleotide group Chemical group 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000010355 oscillation Effects 0.000 description 1
- ISWSIDIOOBJBQZ-UHFFFAOYSA-N phenol group Chemical group C1(=CC=CC=C1)O ISWSIDIOOBJBQZ-UHFFFAOYSA-N 0.000 description 1
- 239000000256 polyoxyethylene sorbitan monolaurate Substances 0.000 description 1
- 235000010486 polyoxyethylene sorbitan monolaurate Nutrition 0.000 description 1
- 108091008146 restriction endonucleases Proteins 0.000 description 1
- 230000007017 scission Effects 0.000 description 1
- 239000011780 sodium chloride Substances 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 238000010257 thawing Methods 0.000 description 1
- GPRLSGONYQIRFK-MNYXATJNSA-N triton Chemical compound [3H+] GPRLSGONYQIRFK-MNYXATJNSA-N 0.000 description 1
- 238000003260 vortexing Methods 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1003—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor
- C12N15/1006—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers
- C12N15/1013—Extracting or separating nucleic acids from biological samples, e.g. pure separation or isolation methods; Conditions, buffers or apparatuses therefor by means of a solid support carrier, e.g. particles, polymers by using magnetic beads
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6806—Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6869—Methods for sequencing
-
- C—CHEMISTRY; METALLURGY
- C40—COMBINATORIAL TECHNOLOGY
- C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
- C40B50/00—Methods of creating libraries, e.g. combinatorial synthesis
- C40B50/06—Biochemical methods, e.g. using enzymes or whole viable microorganisms
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Genetics & Genomics (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biochemistry (AREA)
- Biotechnology (AREA)
- General Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Molecular Biology (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Physics & Mathematics (AREA)
- General Health & Medical Sciences (AREA)
- Biophysics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Plant Pathology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Immunology (AREA)
- General Chemical & Material Sciences (AREA)
- Medicinal Chemistry (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a method for capturing bacterial HI-C genome and plasmid conformation, which comprises the following capturing steps: bacterial culture, cross-linking reaction, sample integrity verification, bacterial lysis, chromatin digestion, biotin filling, connection reaction, reverse cross-linking, library extraction, biotin removal, DNA extraction by phenol chloroform and second-generation library construction. The DNA and the protein are crosslinked together through formaldehyde treatment so as to fix the conformation of the DNA, enzyme digestion, biotin introduction, enzyme linkage and extraction are carried out, then library construction and high-throughput sequencing are carried out on the treated nucleic acid, finally, the interaction information between bacterial chromatin fragments and between the bacterial chromatin fragments and the plasmids can be revealed through analyzing sequencing data, and the method can be applied to genome assembly, gene expression regulation and control research and the like.
Description
Technical Field
The invention relates to the technical field of chromosome conformation capture, in particular to a method for capturing a bacterium HI-C genome and plasmid conformation.
Background
Hi-C mainly comprises the steps of preparing a 3C template suitable for Hi-C, and connecting biotin-labeled nucleotides at the tail end after restriction enzyme cutting. DNA purification cleavage after blunt end ligation. Biotin PulL-down ensures that only the ligated fraction is used for analysis. Sequencing short Reads were mapped to the genome and when the pair of short Reads were found on different fragments, the interacting fragments were scored accordingly. Thus, a ligation frequency matrix is constructed that contains all fragments of the genome. The assay was confirmed by 4C at Hi-C. The Hi-C assay allows scientists to find that chromatin is present in specific chromatin compartments, with chromatin and heterochromatin in distinct regions; human different cell lines, with approximately the same nuclear chromatin organization; bacterial genomic DNA and plasmids also exist in three-dimensional conformations and interactions. In summary, the Hi-C method is a genome-wide, non-limiting, chromatin interaction conformational analysis method based on the "full-on-full" mode using high-throughput sequencing (second or third generation). Relevant findings have been published in both humans and mice. At present, the prior Hi-C chromosome conformation capture technology is not mature and is easy to make mistakes, in particular to a conformation capture method of bacterial genomes and plasmids.
Disclosure of Invention
In order to overcome the defects of the prior technical scheme, the invention provides a bacterial HI-C genome and plasmid conformation capture method, which can effectively solve the problems provided by the background technology.
The technical scheme adopted by the invention for solving the technical problems is as follows:
a method for capturing the conformation of a bacterial HI-C genome and plasmid comprises the following capturing steps:
step S1: and (5) culturing bacteria. Dipping a small amount of bacterial liquid by using an inoculating loop, marking on an LB agar plate, and putting the plate in an incubator at 37 ℃ for culturing for 18-24 hours; selecting single bacteria, and performing shake culture in 50mL LB broth at 37 ℃ for 3 hours and 15 minutes; collecting all bacteria liquid, centrifugally moving at 5000rpm for 8 minutes at 4 ℃, discarding the culture liquid, and collecting bacterial precipitates; add 6mL fresh LB broth, resuspend bacteria, wash; then, carrying out centrifugal motion at 5000rpm for 8 minutes at the temperature of 4 ℃, and collecting bacterial precipitates;
step S2: and (4) carrying out crosslinking reaction. Preparing 20mL of 3% formaldehyde solution, adding the formaldehyde solution into a 50mL centrifuge tube containing the bacterial sediment, re-suspending the bacterial sediment, culturing for 15 minutes at 37 ℃ and shaking every 2 minutes; adding 4.5mL of 2.5M glycine, fully shaking and mixing, then culturing for 5 minutes in a shaking table at 37 ℃, then culturing for 15 minutes in a 4 ℃ environment and shaking every 2 minutes; centrifuging at 5000rpm for 8 min at 4 deg.C, and collecting cross-linked bacteria; adding 4mL of 1 × TE buffer solution, resuspending the bacteria, and then centrifuging at 5000rpm for 8 minutes at 4 ℃; adding 3mL of 1 × TE buffer solution again, re-suspending the bacteria, and evenly distributing the bacteria liquid of the fixed bacteria into a plurality of 2mL of EP tubes; then centrifuged at 5000rpm for 8 minutes at 4 ℃ and the supernatant discarded; rapidly freezing the bacterial precipitate with liquid nitrogen, and storing in a refrigerator at-80 deg.C;
step S3: the sample integrity was verified. Taking one parallel sample out, and extracting DNA by using the kit;
step S4: the bacteria lyse. Taking out the bacteria subjected to the cross-linking reaction from a refrigerator at the temperature of-80 ℃, and putting the bacteria on ice to melt for about 30 minutes; adding 100 mu L of lysozyme into the bacterial sediment, incubating for 15 minutes at the temperature of 30 ℃, and adding 1.5mL of DEPC water to make up the volume;
step S5: chromatin is digested. Preparing a restrictive reaction mixture, adding the mixture into a bacterial lysate, blowing, beating and uniformly mixing, culturing in a water bath solution at 37 ℃ overnight, shaking midway, and immediately placing on ice;
step S6: and (4) biotin filling. Adding the first preparation system into the bacterial digestive juice in the last step, uniformly stirring by blowing, culturing for 2 hours at 25 ℃, and shaking midway; adding an appropriate amount of EDTA (final concentration 10mM) to the mixture, and incubating at 75 ℃ for 20 minutes to inactivate the enzyme; centrifuging at 15000rpm for 30 min at 4 deg.C, removing supernatant, adding 300 μ L DEFC water, and resuspending the precipitate;
step S7: and (3) performing a ligation reaction. Preparing a system II, adding each tube of bacterial liquid in the previous step into a newly prepared system mixture, and turning upside down and uniformly mixing; incubating for 4 hours at 37 ℃ and shaking halfway; then incubating for 10 minutes at 65 ℃ to inactivate the enzyme; finally adding 10 mu L of RNase to incubate for 5 minutes at room temperature;
step S8: and (4) reverse crosslinking. Adding 100 mu L of proteinase K into the ligation product, and incubating for 3 hours at 65 ℃;
step S9: and (4) extracting a library. Adding 1.2 volumes of DNA extracting solution, evenly inverting the upper part and the lower part, and standing for 10 minutes at room temperature; centrifuging at 14000rpm for 10 minutes in an environment of 4 ℃, and rotating the upper layer liquid into a new 15mL centrifugal tube by using a pipette; adding the DNA extracting solution with the volume of 1.2 again, uniformly inverting the solution, and standing the solution at room temperature for 10 minutes; centrifuging at 14000rpm for 10 minutes in an environment of 4 ℃, and rotating the upper layer liquid into a new 15mL centrifugal tube by using a pipette; adding 0.1 volume of 3M sodium acetate and 2 mul of 5.0mg/mL glycogen, and uniformly inverting the mixture up and down; adding 2 volumes of cold 100% ethanol, uniformly turning upside down, and then incubating overnight at-20 ℃; the liquid incubated overnight is filled into 1.5mL centrifuge tubes in batches, the centrifuge tube is centrifuged at 14000rpm for 25 minutes in the environment of 4 ℃, the supernatant fluid is sucked away, and white precipitate exists at the bottom of the centrifuge tube; adding 500 μ L of cold 70% ethanol solution, and washing; centrifuging at 14000rpm for 20 min at 4 deg.C, removing supernatant, and repeating twice; air-drying at room temperature for 20 min; adding 100. mu.L of 1 × TE buffer solution, and dissolving the precipitate; then detecting the DNA concentration by using an HS kit;
step S10: and removing the biotin. Preparing a third system, averagely subpackaging the three systems into PCR tubes, culturing the three systems for 5 minutes in a PCR instrument at the temperature of 12 ℃, wherein each volume of the three systems is 55 mu L; adding 6 μ L EDTA (final concentration is 10mM), and then culturing in a PCR instrument at 75 ℃ for 20 minutes to inactivate the enzyme;
step S11: extracting DNA with phenol chloroform;
step S12: and establishing a library in the second generation.
Further, in step S4, the bacteria were lysed using lysozyme, eliminating the use of a vigorous lysate SDS solution, or using a milder TX-100 lysate, ensuring DNA integrity.
Further, in step S4, the cross-linked bacteria are melted without manual melting; for the bacteria which are difficult to split, 1.5mL of lysate was added and reacted for 5 minutes.
Further, in step S8, high temperature reverse crosslinking is used, the crosslinking time is 3 hours, the high temperature treatment time is reduced, and bacterial DNA degradation is prevented.
Compared with the prior art, the invention has the beneficial effects that:
according to the invention, DNA and protein are crosslinked together through formaldehyde treatment so as to fix DNA conformation, enzyme digestion, biotin introduction, enzyme linkage and extraction are carried out, then library construction and high-throughput sequencing are carried out on the treated nucleic acid, and finally mutual information between bacterial chromatin fragments and plasmids can be revealed through analyzing sequencing data.
Drawings
FIG. 1 is a flow chart of the capture method of the present invention.
Detailed Description
The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the drawings in the embodiments of the present invention, and it is obvious that the described embodiments are only a part of the embodiments of the present invention, and not all of the embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.
As shown in FIG. 1, the present invention provides a method for conformational capture of bacterial HI-C genomes and plasmids, comprising the following capture steps:
step S1: and (5) culturing bacteria. Dipping a small amount of bacterial liquid by using an inoculating loop, marking on an LB agar plate, and putting the plate in an incubator at 37 ℃ for culturing for 18-24 hours; selecting single bacteria, and performing shake culture in 50mL LB broth at 37 ℃ for 3 hours and 15 minutes; collecting all bacteria liquid, centrifugally moving at 5000rpm for 8 minutes at 4 ℃, discarding the culture liquid, and collecting bacterial precipitates; add 6mL fresh LB broth, resuspend bacteria, wash; then, carrying out centrifugal motion at 5000rpm for 8 minutes at the temperature of 4 ℃, and collecting bacterial precipitates;
step S2: and (4) carrying out crosslinking reaction. Preparing 20mL of 3% formaldehyde solution, adding the formaldehyde solution into a 50mL centrifuge tube containing the bacterial sediment, re-suspending the bacterial sediment, culturing for 15 minutes at 37 ℃ and shaking every 2 minutes; adding 4.5mL2.5M glycine, fully shaking and mixing, then culturing for 5 minutes in a shaking table at the temperature of 37 ℃, then culturing for 15 minutes at the temperature of 4 ℃ and shaking every 2 minutes; centrifuging at 5000rpm for 8 min at 4 deg.C, and collecting cross-linked bacteria; adding 4mL1 TE buffer solution, resuspending bacteria, and centrifuging at 5000rpm for 8 min at 4 deg.C; adding 3mL of 1 TE buffer solution again, re-suspending the bacteria, and evenly distributing the bacteria liquid of the fixed bacteria into a plurality of 2mL of EP tubes; then centrifuged at 5000rpm for 8 minutes at 4 ℃ and the supernatant discarded; rapidly freezing the bacterial precipitate with liquid nitrogen, and storing in a refrigerator at-80 deg.C;
step S3: the sample integrity was verified. Taking one parallel sample out, and extracting DNA by using the kit;
step S4: the bacteria lyse. Taking out the bacteria subjected to the cross-linking reaction from a refrigerator at the temperature of-80 ℃, and putting the bacteria on ice to melt for about 30 minutes; adding 100 mu L of lysozyme into the bacterial sediment, incubating for 15 minutes at the temperature of 30 ℃, and adding 1.5mL of DEPC water to make up the volume;
step S5: chromatin is digested. Preparing a restrictive reaction mixture, adding the mixture into a bacterial lysate, blowing, beating and uniformly mixing, culturing in a water bath solution at 37 ℃ overnight, shaking midway, and immediately placing on ice;
step S6: and (4) biotin filling. Adding the first preparation system into the bacterial digestive juice in the last step, uniformly stirring by blowing, culturing for 2 hours at 25 ℃, and shaking midway; adding an appropriate amount of EDTA (final concentration 10mM) to the mixture, and incubating at 75 ℃ for 20 minutes to inactivate the enzyme; centrifuging at 15000rpm for 30 min at 4 deg.C, removing supernatant, adding 300 μ L DEFC water, and resuspending the precipitate;
step S7: and (3) performing a ligation reaction. Preparing a system II, adding each tube of bacterial liquid in the previous step into a newly prepared system mixture, and turning upside down and uniformly mixing; incubating for 4 hours at 37 ℃ and shaking halfway; then incubating for 10 minutes at 65 ℃ to inactivate the enzyme; finally adding 10 mu L of RNase to incubate for 5 minutes at room temperature;
step S8: and (4) reverse crosslinking. Adding 100 mu L of proteinase K into the ligation product, and incubating for 3 hours at 65 ℃;
step S9: and (4) extracting a library. Adding 1.2 volumes of DNA extracting solution, evenly inverting the upper part and the lower part, and standing for 10 minutes at room temperature; centrifuging at 14000rpm for 10 minutes in an environment of 4 ℃, and rotating the upper layer liquid into a new 15mL centrifugal tube by using a pipette; adding the DNA extracting solution with the volume of 1.2 again, uniformly inverting the solution, and standing the solution at room temperature for 10 minutes; centrifuging at 14000rpm for 10 minutes in an environment of 4 ℃, and rotating the upper layer liquid into a new 15mL centrifugal tube by using a pipette; adding 0.1 volume of 3M sodium acetate and 2 mul of 5.0mg/mL glycogen, and uniformly inverting the mixture up and down; adding 2 volumes of cold 100% ethanol, uniformly turning upside down, and then incubating overnight at-20 ℃; the liquid incubated overnight is filled into 1.5mL centrifuge tubes in batches, the centrifuge tube is centrifuged at 14000rpm for 25 minutes in the environment of 4 ℃, the supernatant fluid is sucked away, and white precipitate exists at the bottom of the centrifuge tube; adding 500 μ L of cold 70% ethanol solution, and washing; centrifuging at 14000rpm for 20 min at 4 deg.C, removing supernatant, and repeating twice; air-drying at room temperature for 20 min; adding 100. mu.L of 1 × TE buffer solution, and dissolving the precipitate; then detecting the DNA concentration by using an HS kit;
step S10: and removing the biotin. Preparing a third system, averagely subpackaging the three systems into PCR tubes, culturing the three systems for 5 minutes in a PCR instrument at the temperature of 12 ℃, wherein each volume of the three systems is 55 mu L; adding 6 mu L of EDTA, then culturing for 20 minutes in a PCR instrument at 75 ℃ and inactivating the enzyme;
step S11: extracting DNA with phenol chloroform;
step S12: and establishing a library in the second generation.
In step S4, lysozyme is used for cracking bacteria, so that the use of a violent lysate SDS solution is eliminated, or a milder TX-100 lysate is used, and the integrity of DNA is ensured; in step S4, the cross-linked bacteria are melted without manual melting; in step S8, high temperature reverse crosslinking is used, the crosslinking time is 3 hours, the high temperature treatment time is reduced, and the degradation of bacterial DNA is prevented
Example (b):
the first step is as follows: and (5) culturing bacteria.
1. A small amount of the inoculum was dipped with an inoculating loop, streaked on an LB agar plate (or a tag agar plate), and the plate was incubated in an incubator at 37 ℃ for 18 to 24 hours.
2. The selected bacteria were shake-cultured in 50mL of LB broth at 37 ℃ for 3 hours and 15 minutes.
3. Collecting all bacteria liquid, centrifuging at 4 ℃ and 5000rpm for 8 minutes, discarding the culture solution, and collecting the bacterial precipitate.
4. 6mL of fresh LB broth was added, the bacteria were resuspended and washed. Then, the mixture was centrifuged at 5000rpm for 8 minutes at 4 ℃ to collect bacterial pellets.
The second step is that: and (4) carrying out crosslinking reaction.
1. Prepare 20mL of 3% formaldehyde solution (protected from light on ice): 3% formaldehyde solution
18.4mL of 1 × PBS solution +1.6/mL of 37% w/v formaldehyde solution
2. The prepared 3% formaldehyde solution was added to a 50mL centrifuge tube containing the bacterial pellet, the bacterial pellet was resuspended, incubated at 37 ℃ for 15 minutes, and shaken every 2 minutes.
3. To the crosslinking reaction was added 4.5mL of 2.5M glycine (final concentration of 460mM) and mixed well with shaking. Then incubated at 37 ℃ for 5 minutes with shaking, then at 4 ℃ for 15 minutes, with shaking every 2 minutes.
4. The cross-linked bacteria were collected by centrifugation at 5000rpm for 8 minutes at 4 ℃.
5. 4mL of 1 × TE buffer was added, the bacteria were resuspended, and then centrifuged at 5000rpm for 8 minutes at 4 ℃.
6. 3mL of 1 × TE buffer was added again to resuspend the bacteria, and the bacteria-fixed suspension was equally divided into 2mL EP tubes, 1mL per tube (three in parallel).
7. Then, the mixture was centrifuged at 5000rpm for 8 minutes at 4 ℃ and the supernatant was discarded.
8. The bacterial pellets were rapidly frozen using liquid nitrogen and stored in a freezer at-80 ℃.
The third step: the sample integrity was verified.
One of the replicates was removed and the kit extracted DNA to ensure bacterial DNA integrity. (bacterial DNA integrity to be injected into Hi-C library construction)
The fourth step: the bacteria lyse.
1. The cross-linked bacteria were removed from the-80 ℃ freezer and placed on ice to melt for about 30 minutes without manual thawing.
2. To the bacterial pellet, 100. mu.L of lysozyme was added and incubated at 30 ℃ for 15 minutes.
3. 1.5mL of DEPC water was added to make up the volume.
(for bacteria that are difficult to rupture, 1.5mL of lysate is added for 5 minutes)
The fifth step: chromatin is digested.
(working on ice) preparation of a limiting reaction mixture:
| HindIII | 20000U/mL | 10μL |
| Buffer | 50μL |
2. the restriction reaction mixture was added to the bacterial lysate, pipetted and mixed, incubated overnight in a 37 ℃ water bath, shaken once in the middle.
3. And then immediately placed on ice.
And a sixth step: and (4) biotin filling.
1. The following systems were prepared in order:
| DEFC water | 17.1μL |
| NE Buffer | 16.4μL |
| 10mM dCTP | 3μL |
| 10mM dGTP | 3μL |
| 10mM dTTP | 3μL |
| 0.4mM biotin-14-dATP | 7.5μL |
| 5U/. mu.L DNA polymerase I Klenow | 10μL |
2. Adding the system into the bacterial digestive juice in the last step, and uniformly mixing by blowing. Incubated at 25 ℃ for 2 hours with shaking halfway.
3. The enzyme was inactivated by adding an appropriate amount of EDTA (final concentration 10mM) to the mixture and incubating at 75 ℃ for 20 minutes.
4. Centrifuge at 15000rpm for 30 minutes at 4 ℃. The supernatant was removed, 300. mu.L DEFC water was added, and the pellet was resuspended.
The seventh step: and (3) performing a ligation reaction.
(on ice procedure) the following system was formulated in a new 2.0mL centrifuge tube:
| Nuclease-free water | 661μL |
| 10x T4 ligation buffer | 120μL |
| 10％Triton X-10 | 100μL |
| 10mg/mL BSA | 14μL |
| T4 DNA ligase(2000U/μL)NEB | 5μL |
adding each tube of the bacterial solution in the previous step into the prepared mixture, and turning upside down and mixing uniformly.
2. Incubate at 37 ℃ for 4 hours with shaking.
Incubate at 3.65 ℃ for 10 minutes, inactivate the enzyme.
4. Add 10. mu.L RNase and incubate for 5 min at room temperature.
Eighth step: and (4) reverse crosslinking.
To the ligation product, 100. mu.L of proteinase K (20mg/mL) was added and incubated at 65 ℃ for 3 hours.
The ninth step: and (4) extracting a library.
1. The DNA extract solution was added in an amount of 1.2 volumes, mixed by gently inverting it upside down, and allowed to stand at room temperature for 10 minutes.
2. Centrifuge at 14000rpm for 10 minutes at 4 ℃ and transfer the supernatant to a new 15mL centrifuge tube using a pipette. (liquid layering, lower layer is phenol phase, middle white layer is denatured protein, upper layer is water phase containing DNA)
3. The DNA extract solution was added again in a volume of 1.2 volumes, mixed by gently inverting the mixture upside down, and allowed to stand at room temperature for 10 minutes.
4. Centrifuge at 14000rpm for 10 minutes at 4 ℃ and transfer the supernatant to a new 15mL centrifuge tube using a pipette.
5. 0.1 volume of 3M sodium acetate and 2. mu.L of 5.0mg/mL glycogen were added, and the mixture was mixed by inverting the mixture upside down.
6. 2 volumes of cold 100% ethanol were added and mixed by inversion.
7. Followed by incubation at-20 ℃ overnight.
8. The overnight incubated liquid was aliquoted into 1.5mL centrifuge tubes and centrifuged at 14000rpm for 25 minutes at 4 ℃. The supernatant was aspirated off and a white precipitate was seen at the bottom of the centrifuge tube.
9. Add 500. mu.L of cold 70% ethanol solution and wash (do not suspend the pellet).
10. Centrifugation was carried out at 14000rpm for 20 minutes at 4 ℃ to remove the supernatant, and this was repeated twice.
11. Air-dry at room temperature for 20 minutes.
12. Add 100. mu.L of 1 × TE buffer and dissolve the pellet.
13. The DNA concentration was detected with HS kit.
The tenth step: and removing the biotin.
(ice operation) preparing a system according to the following proportion, and uniformly mixing:
2. each tube was divided into PCR tubes in an average of 55. mu.L each. Incubate at 12 ℃ for 5 minutes in a PCR instrument.
3. mu.L of EDTA was added, and the mixture was incubated at 75 ℃ in a PCR apparatus for 20 minutes to inactivate the enzyme.
The eleventh step: phenol chloroform extraction of DNA.
Finally, the pellet was dissolved in 30. mu.L of 1 × TE buffer.
The twelfth step: and establishing a library in the second generation.
(one) enzyme digestion reaction
1. Preparing enzyme digestion PCR system
mu.L Buffer was added to 16. mu.L of the DNA product, 2. mu.L of the enzyme was added, and the mixture was pipetted and mixed.
PCR procedure:
5 minutes at 37 DEG C
After PCR, the sample was inserted on ice, 5. mu.L of EDTA was immediately added, and the mixture was blown up and mixed.
4. 5. mu.L of the solution was aspirated for an agarose gel electrophoresis experiment.
5. Purification was performed using 2.2X magnetic beads.
6. The DNA concentration was detected using HS.
(II) end repair
1. The terminal buffer and terminal repair enzyme (no vortexing, low speed centrifugation) were removed from the freezer and placed on ice.
PCR System preparation (50. mu.L)
PCR procedure:
(III) linker reaction
1. The adaptor, ligase, ligation Buffer2-1 (Buffer2-1) were removed from the freezer and placed on ice. The enhancing solution was removed from the room temperature and Buffer2-2 (Buffer2-2) was ligated.
PCR System preparation (80. mu.L):
PCR procedure:
(IV) streptavidin magnetic bead preparation
1. Preparing 100mL Buffer I:
| 10mM Tris-HCl(pH7.5) | 0.1211g |
| 1mM EDTA | 0.0372g |
| 1M NaCl | 0.0058g |
| 0.05％ Tween-20 | 50μL |
2. and (3) fully suspending the magnetic beads, placing the mixture on a mixer, carrying out vortex oscillation for 20 seconds, taking 50 mu L of the magnetic beads to a new centrifugal tube, placing the centrifugal tube on a magnetic frame, carrying out magnetic separation, and discarding supernatant.
3. Add 200 u L Buffer I, fully washing magnetic beads.
And (3) magnetic bead washing process: add buffer to PCR tube, cover the tube cap, vortex the beads for 30 seconds, magnetically separate, discard the supernatant. Repeat the above step 1 time.
4. Add 120. mu.L of Buffer I and resuspend the beads.
5. Add 80. mu.L of linker reaction product to the beads and mix them well by pipetting. Incubate at 37 ℃ for 45 minutes.
6. The mixture was placed on a magnetic stand and allowed to stand for 10 minutes (magnetic separation). The supernatant was removed to a new centrifuge tube for use.
7. Add 200 u L Buffer I, fully washing magnetic beads. Repeat for 1 time.
8. Add 23. mu.L of DEPC water and resuspend the beads.
(V) library amplification
PCR System:
PCR procedure:
3. the PCR tube was placed on a magnetic stand and allowed to stand for 5 minutes. Then transfer 48. mu.L of the supernatant to a new PCR tube, put on ice until used again.
4. Washing with the remaining magnetic beads
(1) Add 50. mu.L of Buffer I into the PCR tube containing the magnetic beads in the previous step, and blow and mix them well. The PCR tube was placed on a magnetic stand, allowed to stand for 5 minutes, and then the supernatant was taken out. (repeat twice)
(2) Add 50. mu.L of TE buffer to the PCR tube containing the beads in the previous step, resuspend the beads, and store at 4 ℃. (Can continue to use)
(VI) library purification
1. Pipet 48. mu.L of the magnetic bead to 48. mu.L of the supernatant, and mix them by pipetting. The mixture was allowed to stand at room temperature for 5 minutes.
2. The mixture was placed in a magnetic stand for 5 minutes. Add 200. mu.L of 80% ethanol, let stand for 30 seconds, and discard the liquid. And repeating the steps once.
3. After leaving at room temperature for 5 minutes, the magnetic beads were dried to volatilize the alcohol.
4. Add 25. mu.L of ddH2O, resuspend the beads, pipette well, and let stand at room temperature for 5 minutes.
5. Placing the mixture on a magnetic frame and standing for 5 minutes.
6. Aspirate 23. mu.L of supernatant into a new PCR tube and aspirate 2. mu.L of supernatant for HS detection of DNA concentration.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Claims (4)
1. The method for capturing the conformation of the HI-C genome and plasmid of the bacteria is characterized by comprising the following capturing steps:
step S1: and (5) culturing bacteria. Dipping a small amount of bacterial liquid by using an inoculating loop, marking on an LB agar plate, and putting the plate in an incubator at 37 ℃ for culturing for 18-24 hours; selecting single bacteria, and performing shake culture in 50mL LB broth at 37 ℃ for 3 hours and 15 minutes; collecting all bacteria liquid, centrifugally moving at 5000rpm for 8 minutes at 4 ℃, discarding the culture liquid, and collecting bacterial precipitates; add 6mL fresh LB broth, resuspend bacteria, wash; then, the bacterial pellet was collected by centrifugal movement at 5000rpm for 8 minutes under an environment of 4 ℃.
Step S2: and (4) carrying out crosslinking reaction. Preparing 20mL of 3% formaldehyde solution, adding the formaldehyde solution into a 50mL centrifuge tube containing the bacterial sediment, re-suspending the bacterial sediment, culturing for 15 minutes at 37 ℃ and shaking every 2 minutes; adding 4.5mL2.5M glycine, fully shaking and mixing, then culturing for 5 minutes in a shaking table at the temperature of 37 ℃, then culturing for 15 minutes at the temperature of 4 ℃ and shaking every 2 minutes; centrifuging at 5000rpm for 8 min at 4 deg.C, and collecting cross-linked bacteria; adding 4mL1 TE buffer solution, resuspending bacteria, and centrifuging at 5000rpm for 8 min at 4 deg.C; adding 3mL of 1 TE buffer solution again, re-suspending the bacteria, and evenly distributing the bacteria liquid of the fixed bacteria into a plurality of 2mL of EP tubes; then centrifuged at 5000rpm for 8 minutes at 4 ℃ and the supernatant discarded; the bacterial pellets were rapidly frozen using liquid nitrogen and stored in a freezer at-80 ℃.
Step S3: the sample integrity was verified. One of the replicates was removed and the kit was used to extract DNA.
Step S4: the bacteria lyse. Taking out the bacteria subjected to the cross-linking reaction from a refrigerator at the temperature of-80 ℃, and putting the bacteria on ice to melt for about 30 minutes; to the bacterial pellet was added 100. mu.L lysozyme, incubated at 30 ℃ for 15 minutes and 1.5mL DEPC water was added to make up the volume.
Step S5: chromatin is digested. A restriction reaction mixture was prepared and added to the bacterial lysate, blown up and mixed well, incubated overnight in a 37 ℃ water bath, shaken midway through and then immediately placed on ice.
Step S6: and (4) biotin filling. Adding the first preparation system into the bacterial digestive juice in the last step, uniformly stirring by blowing, culturing for 2 hours at 25 ℃, and shaking midway; adding an appropriate amount of EDTA (final concentration 10mM) to the mixture, and incubating at 75 ℃ for 20 minutes to inactivate the enzyme; after centrifugation at 15000rpm for 30 minutes at 4 ℃ for 30 minutes, the supernatant was removed, 300. mu.L of DEFC water was added, and the pellet was resuspended.
Step S7: and (3) performing a ligation reaction. Preparing a system II, adding each tube of bacterial liquid in the previous step into a newly prepared system mixture, and turning upside down and uniformly mixing; incubating for 4 hours at 37 ℃ and shaking halfway; then incubating for 10 minutes at 65 ℃ to inactivate the enzyme; finally, 10. mu.L of RNase was added and incubated at room temperature for 5 minutes.
Step S8: and (4) reverse crosslinking. To the ligation product, 100. mu.L of proteinase K was added and incubated at 65 ℃ for 3 hours.
Step S9: and (4) extracting a library. Adding 1.2 volumes of DNA extracting solution, evenly inverting the upper part and the lower part, and standing for 10 minutes at room temperature; centrifuging at 14000rpm for 10 minutes in an environment of 4 ℃, and rotating the upper layer liquid into a new 15mL centrifugal tube by using a pipette; adding the DNA extracting solution with the volume of 1.2 again, uniformly inverting the solution, and standing the solution at room temperature for 10 minutes; centrifuging at 14000rpm for 10 minutes in an environment of 4 ℃, and rotating the upper layer liquid into a new 15mL centrifugal tube by using a pipette; adding 0.1 volume of 3M sodium acetate and 2 mu L5.0mg/mL glycogen, and uniformly inverting the mixture from top to bottom; adding 2 volumes of cold 100% ethanol, uniformly turning upside down, and then incubating overnight at-20 ℃; the liquid incubated overnight is filled into 1.5mL centrifuge tubes in batches, the centrifuge tube is centrifuged at 14000rpm for 25 minutes in the environment of 4 ℃, the supernatant fluid is sucked away, and white precipitate exists at the bottom of the centrifuge tube; adding 500 μ L of cold 70% ethanol solution, and washing; centrifuging at 14000rpm for 20 min at 4 deg.C, removing supernatant, and repeating twice; air-drying at room temperature for 20 min; adding 100. mu.L of 1 × TE buffer solution, and dissolving the precipitate; and then detecting the DNA concentration by using an HS kit.
Step S10: and removing the biotin. Preparing a third system, averagely subpackaging the three systems into PCR tubes, culturing the three systems for 5 minutes in a PCR instrument at the temperature of 12 ℃, wherein each volume of the three systems is 55 mu L; mu.L of EDTA (10 mM final concentration) was added, and the mixture was incubated in a PCR apparatus at 75 ℃ for 20 minutes to inactivate the enzyme.
Step S11: phenol chloroform extraction of DNA.
Step S12: and establishing a library in the second generation.
2. The method of claim 1, wherein the method of conformational capture of bacterial HI-C genomes and plasmids comprises: in step S4, the bacteria were lysed using lysozyme, eliminating the use of a vigorous lysate SDS solution, or using a milder TX-100 lysate, ensuring DNA integrity.
3. The method of claim 1, wherein the method of conformational capture of bacterial HI-C genomes and plasmids comprises: in step S4, the cross-linked bacteria are melted without manual melting; for the bacteria which are difficult to split, 1.5mL of lysate was added and reacted for 5 minutes.
4. The method of claim 1, wherein the method of conformational capture of bacterial HI-C genomes and plasmids comprises: in step S8, high temperature reverse crosslinking is used, with a crosslinking time of 3 hours, reducing the high temperature processing time and preventing bacterial DNA degradation.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110204762.3A CN113215141A (en) | 2021-02-23 | 2021-02-23 | Bacterial HI-C genome and plasmid conformation capture method |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110204762.3A CN113215141A (en) | 2021-02-23 | 2021-02-23 | Bacterial HI-C genome and plasmid conformation capture method |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN113215141A true CN113215141A (en) | 2021-08-06 |
Family
ID=77084685
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110204762.3A Pending CN113215141A (en) | 2021-02-23 | 2021-02-23 | Bacterial HI-C genome and plasmid conformation capture method |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN113215141A (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116606910A (en) * | 2023-07-21 | 2023-08-18 | 中国农业科学院农业基因组研究所 | Metagenomic GutHi-C library building method suitable for microbial population and application |
Citations (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014121091A1 (en) * | 2013-02-01 | 2014-08-07 | The Regents Of The University Of California | Methods for genome assembly and haplotype phasing |
| CN105992825A (en) * | 2013-11-18 | 2016-10-05 | 鹿特丹伊拉斯谟大学医疗中心 | Method for analysing the interaction of nucleotide sequences in a three-dimensional dna structure |
| WO2017066907A1 (en) * | 2015-10-19 | 2017-04-27 | 安诺优达基因科技(北京)有限公司 | Method of building hi-c library having high usable data ratio |
| CN109056078A (en) * | 2018-09-18 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of Hi-C high-flux sequence banking process suitable for bacterium |
| WO2019071054A1 (en) * | 2017-10-04 | 2019-04-11 | The Broad Institute, Inc. | Methods and compositions for altering function and structure of chromatin loops and/or domains |
| US20190385703A1 (en) * | 2016-06-08 | 2019-12-19 | The Broad Institute, Inc. | Linear genome assembly from three dimensional genome structure |
| CN111445948A (en) * | 2020-03-27 | 2020-07-24 | 武汉古奥基因科技有限公司 | Chromosome construction method for polyploid fish by using Hi-C |
| WO2020161485A1 (en) * | 2019-02-05 | 2020-08-13 | Oxford University Innovation Limited | Process for producing a chromatin conformation capture (3c) library |
| CN111909983A (en) * | 2020-08-25 | 2020-11-10 | 武汉菲沙基因信息有限公司 | Hi-C high-throughput sequencing database building method applicable to microbial metagenomics and application |
-
2021
- 2021-02-23 CN CN202110204762.3A patent/CN113215141A/en active Pending
Patent Citations (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2014121091A1 (en) * | 2013-02-01 | 2014-08-07 | The Regents Of The University Of California | Methods for genome assembly and haplotype phasing |
| CN105121661A (en) * | 2013-02-01 | 2015-12-02 | 加利福尼亚大学董事会 | Methods for genome assembly and haplotype phasing |
| CN105992825A (en) * | 2013-11-18 | 2016-10-05 | 鹿特丹伊拉斯谟大学医疗中心 | Method for analysing the interaction of nucleotide sequences in a three-dimensional dna structure |
| WO2017066907A1 (en) * | 2015-10-19 | 2017-04-27 | 安诺优达基因科技(北京)有限公司 | Method of building hi-c library having high usable data ratio |
| US20190385703A1 (en) * | 2016-06-08 | 2019-12-19 | The Broad Institute, Inc. | Linear genome assembly from three dimensional genome structure |
| WO2019071054A1 (en) * | 2017-10-04 | 2019-04-11 | The Broad Institute, Inc. | Methods and compositions for altering function and structure of chromatin loops and/or domains |
| US20200255828A1 (en) * | 2017-10-04 | 2020-08-13 | The Broad Institute, Inc. | Methods and compositions for altering function and structure of chromatin loops and/or domains |
| CN109056078A (en) * | 2018-09-18 | 2018-12-21 | 武汉菲沙基因信息有限公司 | A kind of Hi-C high-flux sequence banking process suitable for bacterium |
| WO2020161485A1 (en) * | 2019-02-05 | 2020-08-13 | Oxford University Innovation Limited | Process for producing a chromatin conformation capture (3c) library |
| CN111445948A (en) * | 2020-03-27 | 2020-07-24 | 武汉古奥基因科技有限公司 | Chromosome construction method for polyploid fish by using Hi-C |
| CN111909983A (en) * | 2020-08-25 | 2020-11-10 | 武汉菲沙基因信息有限公司 | Hi-C high-throughput sequencing database building method applicable to microbial metagenomics and application |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116606910A (en) * | 2023-07-21 | 2023-08-18 | 中国农业科学院农业基因组研究所 | Metagenomic GutHi-C library building method suitable for microbial population and application |
| CN116606910B (en) * | 2023-07-21 | 2023-10-13 | 中国农业科学院农业基因组研究所 | Metagenomic GutHi-C library building method suitable for microbial population and application |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP6324962B2 (en) | Methods and kits for preparing target RNA depleted compositions | |
| CN109797436B (en) | Sequencing library construction method | |
| Rhee et al. | ChIP‐exo method for identifying genomic location of DNA‐binding proteins with near‐single‐nucleotide accuracy | |
| CN108624651B (en) | Method for constructing Ribo-seq sequencing library | |
| CN111440896A (en) | Novel β coronavirus variation detection method, probe and kit | |
| CN107058295A (en) | A kind of whole blood DNA rapid extracting method | |
| CN111778563A (en) | Construction method of cell Hi-C sequencing library | |
| CN111909983A (en) | Hi-C high-throughput sequencing database building method applicable to microbial metagenomics and application | |
| CN113215141A (en) | Bacterial HI-C genome and plasmid conformation capture method | |
| CN110885845A (en) | Method for screening active enhancer by using vector based on random sequence | |
| Ribaudo et al. | Preparation of RNA from tissues and cells | |
| US5096818A (en) | Nucleic acid separation method | |
| CN106754868A (en) | A kind of method of the DNA fragmentation interacted in capture Matrix attachment region | |
| CN112481254B (en) | Method and kit for removing host DNA and enriching microorganisms by one-step method | |
| CN111607588A (en) | Annular RNA rapid enrichment kit and method | |
| CN115820807B (en) | Long fragment capturing and enriching method, kit and application thereof | |
| CN115109836A (en) | Hi-C detection method based on flora | |
| CN114703173B (en) | Lambda phage DNA extraction kit and extraction method | |
| CN116262918A (en) | Probe set, kit and application thereof, method for removing ribosomal RNA in sample and method for extracting nucleic acid in biological sample | |
| WO1998016653A1 (en) | Compositions and methods for rapid isolation of plasmid dna | |
| CN107988317A (en) | A kind of buffer solution that hybrid capture is carried out to target set nucleic acid region | |
| Moore | Purification and concentration of DNA from aqueous solutions | |
| CN113249376A (en) | Method for capturing melon mitochondrial genome based on CRISPR-Cas and application of method | |
| CN115279917A (en) | Method for multidimensional cell epigenomics analysis | |
| CN111455021B (en) | Method and kit for removing host DNA in metagenome |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| RJ01 | Rejection of invention patent application after publication | ||
| RJ01 | Rejection of invention patent application after publication |
Application publication date: 20210806 |