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
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[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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