Hi-C high-throughput sequencing library construction method applicable to plants
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A high-throughput, plant technology, applied in the field of molecular biology, can solve the problems of poor library construction effect of difficult-to-release cytoplasmic plant samples, and achieve the effects of improving integrity and cross-linking effect, shortening reaction time, and simplifying operation steps.
Inactive Publication Date: 2018-12-21
WUHAN FRASERGEN CO LTD
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[0004] Aiming at the technical problems existing in the prior art, the present invention proposes a Hi-C high-throughput sequencing library construction method suitable for plants, which is designed by designing suitable formaldehyde cross-linking conditions, endogenous enzyme inactivation conditions and suitable The tool enzyme solves the technical problems of difficult release of cytoplasm from plant cells and poor quality of plant sample library construction, realizes Hi-C high-throughput sequencing library construction of plants, and expands the scope of application of Hi-C technology
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Embodiment 1
[0051] In this example, healthy soybean leaves were taken as the research object. Fresh leaves were cross-linked with formaldehyde, cells were lysed to release chromatin, and then the chromatin was digested, biotin-labeled, intramolecularly connected, and ultrasonically interrupted, and finally the library was constructed. The specific experiment The process is as follows:
[0052] 1. Formaldehyde fixation
[0053] 1) Take 1 g of young soybean leaves (preferably without a waxy layer), cut them into pieces about 0.5 cm in size, put them into a 50 mL centrifuge tube, and add 35 mL of pre-cooled NIB lysate to the centrifuge tube (add 35 μL of mercapto ethanol and 35 μL of 100 mM PMSF) and 3 mL of about 37% formaldehyde solution (final concentration 2%), vacuumize on ice for 40 min;
[0054] 2) Add 7.5mL 2.0M glycine and treat under vacuum for 5min to terminate cross-linking;
[0055] 3) Remove the solution and wash 2-3 times with sterilized ultrapure water, and completely dry t...
Embodiment 2
[0108] In this example, soybean leaves with diseases and insect pests were taken as the research object. The soybean leaves with disease and insect pests were cross-linked with formaldehyde, the cells were lysed to release chromatin, and then the chromatin was digested, biotin-labeled, intramolecularly linked, and ultrasonically interrupted, and finally the library was constructed. The experimental process is the same as in Example 1. Identify the genome integrity, enzyme digestion effect, and connection effect by agarose gel electrophoresis pattern. The agarose gel electrophoresis pattern is as follows: Figure 4 As shown in the picture, the complete genomic DNA bands of soybean leaves affected by diseases and insect pests are obviously without tailing; the enzymatic digestion effect is obvious, and the entire DNA fragment moves down; Proceed to the next experiment.
[0109] For the clean data obtained by quality control, use ICE3 software to iteratively compare the data and...
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Abstract
The invention relates to a Hi-C high-throughput sequencing library construction method applicable to plants. The method comprises the following steps: (1) performing vacuum formaldehyde cross-linkingon sample ice; (2) cracking the cross-linked material, and releasing cell chromatin; performing endogenous enzyme inactivation on the cell chromatin by SDS (Sodium Dodecyl Sulfate), performing enzymedigestion cutting by MboI endonuclease to obtain a material subjected to enzyme digestion, wherein the concentration of the MboI endonuclease is 5000 units / mL, and the enzyme digestion condition is that the material is incubated overnight at 37 DEG C at 900 revolutions per minute; (3) performing end repair on the material subjected to enzyme digestion; (4) performing DNA intramolecular ligation after end repair; (5) decrosslinking the material subjected to intramolecular ligation, removing the non-ligation terminal biotin to obtain purified DNA, randomly breaking the DNA into fragments favorable for sequencing by ultrasonic waves, so as to obtain the DNA sequencing library. According to the method, by designing the appropriate formaldehyde cross-linking conditions, endogenous enzyme inactivation conditions and appropriate tool enzymes, the technical problems that the cytoplasm is difficultly released from plant cells and the library construction effect of low-quality plant samples is poor are solved, the Hi-C high-throughput sequencing library construction of the plants is realized, and the application range of the Hi-C technology is widened.
Description
technical field [0001] The present invention relates to the technical field of molecular biology, and more specifically relates to a Hi-C high-throughput sequencing library construction method suitable for plants. Background technique [0002] Chromosome Conformation Capture (3C) technology is a technique for studying chromosome-protein interaction and chromosome conformation, which can provide detailed information on the association between distant genetic loci, which can be obtained from formaldehyde captured in the nucleus and can be deduced from the three-dimensional folding of chromosomes. In recent years, with the rapid development of second-generation sequencing technology, Hi-C derived from 3C technology takes the entire nucleus as the research object to study the correlation between gene loci in the entire genome. In Hi-C technology, the entire cell line is used as the research object, using high-throughput sequencing technology, combined with bioinformatics method...
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