A simple and effective method for constructing plant long-fragment in situ DLO Hi-C sequencing library

A technology of sequencing library and construction method, which is applied in the field of construction of plant long-fragment insituDLOHi-C sequencing library, can solve the problems of sequencing cost and resolution limitation, and achieves the improvement of genome alignment rate, good integrity, and avoidance of completeness. Effect

Active Publication Date: 2021-09-07
HUAZHONG AGRI UNIV
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  • Description
  • Claims
  • Application Information

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Problems solved by technology

Using this method, scientists have done more work on species with smaller genomes such as Arabidopsis and rice, but due to the limitation of sequencing cost and resolution, it is difficult to do a good job in plant species with large genomes. Perform higher resolution TADs and loops studies

Method used

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  • A simple and effective method for constructing plant long-fragment in situ DLO Hi-C sequencing library
  • A simple and effective method for constructing plant long-fragment in situ DLO Hi-C sequencing library
  • A simple and effective method for constructing plant long-fragment in situ DLO Hi-C sequencing library

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preparation example Construction

[0059] The preparation method of the linker comprises: after the pre-sense strand and the post-sense strand are hybridized to obtain the linker, the nucleotide sequence of the pre-sense strand is shown in SEQ ID No.1, and the nucleotide sequence of the post-sense strand is The sequence is shown as SEQ ID No.2;

[0060] 6) centrifuging and rinsing the linker connector obtained in step 5) to obtain a cell nucleus, mixing the cell nucleus with the phosphorylation system at the end of the linker, and bathing in water at 37° C. for 25 to 35 minutes to obtain a second water bath;

[0061] The linker terminal phosphorylation system component is 170μl ddH 2 O, 20 μl of 10×T4 ligase buffer, 10 μl of T4 polynucleotide kinase with enzyme activity of 10 unit / μl and 5 μl of Triton X-100 solution with a concentration of 20% by volume;

[0062] 7) After mixing the second water bath in step 6) with the connection system, at 20-25°C, connect the chromatin in the nucleus for 1.5-3 hours to obt...

Embodiment 1

[0101] 1. Take freshly harvested 2-4mm long tassels and female ears of maize at the development stage as tissue samples, carry out 2 biological repetitions respectively, and totally 4 complete experiments, named as tassel 1, tassel 2, Ear 1 and ear 2, and the same experimental procedures were used in the four experiments to verify the stability and reliability of the method.

[0102] 2. Put the tissue in an EP tube of appropriate size, add formaldehyde cross-linking buffer (1×PBS containing 1% sigma formaldehyde), fix under vacuum for 15 min for cross-linking, and add glycine to a concentration of 0.2M to stop cross-linking. Immediately afterwards, the plant tissue was rinsed three times with double distilled water, and directly proceeded to the next step or frozen at -80°C for storage.

[0103] 3. Take 0.1-2g of the cross-linked tissue sample and place it in a plate containing PP buffer (1×PBS containing protease inhibitors) pre-cooled on ice, and mash the tissue as much as p...

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Abstract

The invention provides a simple and effective method for constructing plant long fragment in situ DLO Hi-C sequencing library, which belongs to the technical field of plant three-dimensional genome. Mix with Triton X-100, centrifuge, mix the cell nucleus with the system, digest, connect the digested product with the linker, centrifuge, resuspend the cell nucleus in the system, mix with the connection system after a water bath, and centrifuge, and dissociate the cell nucleus Mix the linked system, uncrosslink, purify, incubate the DNA with the pretreated streptavidin magnetic beads, mix the magnetic beads with the Tn5 transposase system, digest and add adapters, wash away the background, amplify, and enrich DNA fragments to obtain sequencing libraries. When the method provided by the invention is used as the test object, the high-throughput data generated has strong advantages in data signal-to-noise ratio and chromatin interaction identification.

Description

technical field [0001] The invention belongs to the field of plant three-dimensional genome technology, and in particular relates to a simple and effective method for constructing a plant long fragment insitu DLO Hi-C sequencing library. Background technique [0002] As the carrier of eukaryotic genetic information, chromatin is not randomly distributed in the nucleus, and its spatial organization plays an important role in DNA replication, DNA damage repair and gene transcription regulation. 3C (chromatin conformation capture) technology was first introduced in 2002 to explore long-range chromatin interactions. This method analyzes the interaction strength of a pair of specific genomic loci (one-to-one) based on the spatial proximity of chromatin. On the basis of 3C technology, including 4C (one locus to all loci) and 5C (many to many) techniques were developed to study genome structure and chromatin interaction. With the development of high-throughput sequencing technolo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C40B50/06
CPCC40B50/06
Inventor 杨芳孙永浩林达曹罡
Owner HUAZHONG AGRI UNIV
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