Lysate for extracting plant cell nucleus

A lysate and cell nucleus technology, which is used in the extraction of plant cell nucleus and the lysate field of plant ATAC-seq experiments. It can solve the problems of different tissue characteristics and inappropriate plant cell nucleus extraction, and achieve the effect of complete nuclear membrane and high sequencing quality.

Pending Publication Date: 2022-04-05
THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] Since plant cells have cell walls, and their cell membranes and nuclear membranes are different from animal cells, 10x Genomics' lysate is not suitable for plant nucleus extraction
[0008] Moreover, the tissue characteristics of different parts of the plant, different developmental stages, and different environmental conditions are different, and the same concentration of lysate is not suitable for different tissues.

Method used

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  • Lysate for extracting plant cell nucleus
  • Lysate for extracting plant cell nucleus
  • Lysate for extracting plant cell nucleus

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0050] Example 1 Carry out rice root tip nucleus extraction with the lysate developed by the present invention

[0051] 1. Purpose of the experiment

[0052] Test the effect of the lysate provided by the invention in extracting rice root tip nuclei.

[0053] 2. Experimental materials

[0054] 1, paddy rice root tip (comprising radicle and crown root), is cultivated according to the following method:

[0055] The rice seeds were germinated at 37°C for 2 days, planted, 14 hours of darkness, 10 hours of light, and the light intensity was 10,000lx. (When the radicle needs to be cultivated, it is cultivated for 3 days; if it is necessary to obtain crown roots, it is cultivated for 6 days). The culture medium is Hogland nutrient solution. After the rice root grows, a 5mm part of the root tip is taken for cell nucleus extraction.

[0056] 2. Lysis solution

[0057] First prepare the base solution according to Table 1

[0058] Table 1 The amount of each component in the base flu...

Embodiment 2

[0106] Example 2 Use the lysate developed by the present invention to extract rice root tip nuclei under stress conditions (this example is heat stress)

[0107] 1. Purpose of the experiment

[0108] The lysate extracted from rice cell nuclei under different stress conditions (such as heat stress, cold stress, salt stress, drought stress, etc.) was developed.

[0109] The heat-stressed rice root tip lysate developed by the present invention is tested for the effect of extracting heat-stressed rice root tip nuclei.

[0110] 2. Experimental materials

[0111] 1, paddy rice root tip (comprising radicle and crown root), is cultivated according to the following method:

[0112] The rice seeds were germinated at 37°C for 2 days, planted at 10:00, 14 hours of darkness, 10 hours of light, and the light intensity was 10,000lx. (When the radicle needs to be cultivated, it is cultivated for 3 days; if it is necessary to obtain crown roots, it is cultivated for 6 days), The culture med...

Embodiment 3

[0128] Example 3 Use the lysate developed by the present invention to extract the nuclei of other rice tissues

[0129] 1. Purpose of the experiment

[0130] Preparation of lysates for cell nuclei extraction from different rice tissues.

[0131] The effect of extracting rice young panicle nuclei by the rice young panicle lysate developed by the present invention was tested.

[0132] 2. Experimental materials

[0133] 1. The young ears of rice are cultivated according to the following methods:

[0134] The rice seeds were planted in an outdoor field in Beijing in May, and when the young panicle grew to 0.5-1 cm long, it was taken out for cell nucleus extraction.

[0135] 2. Lysis solution

[0136] First prepare the base solution according to Table 3

[0137] Table 3 The dosage of each component in the base fluid

[0138]

[0139] Base liquid preparation method:

[0140] Add enzyme-free sterile water to the centrifuge tube first, and then add the ingredients in the abo...

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Abstract

The invention provides a lysate which is used for plant cell nucleus extraction and ATAC-seq experiments based on a lysate of 10x Genomics Company. Under the condition that the basic components of the original formula are not changed as far as possible, the proper concentration of each component is found, and cell nucleuses of different tissue parts, development periods and stress states of plants can be extracted. It is found for the first time that the fixed time needs to be adjusted according to species, tissue parts, plant development periods and environmental states (such as stress of heat, cold, salt, drought and the like). It is found for the first time that extraction is carried out after plant materials are fixed, and beta-mercaptoethanol is added into original lysate components, so that plant cell nucleus extraction and plant ATAC-seq experiments can be carried out. Experiments prove that when the lysate provided by the invention is used for extracting the plant cell nucleus, the nuclear membrane of the obtained cell nucleus is complete, and the sequencing quality is high; the method is suitable for different tissue parts, different development periods and different environment states of different plant species.

Description

Technical field: [0001] The invention belongs to the field of biotechnology, and in particular relates to a lysate for extracting plant nuclei and plant ATAC-seq experiments. Background technique: [0002] The state of chromatin in the nucleus changes continuously with developmental stages or external stimuli. In order to reveal the epigenetic mechanism related to specific cellular processes and stress states, chromatin accessibility analysis and sequencing (ATAC-seq, Assay for Transposase- Accessible Chromatin using sequencing). ATAC-seq requires higher purity nuclei. [0003] The nucleus of higher animals is surrounded by the nuclear membrane and located inside the cell, outside of which there are cytoplasm and various organelles, and the outermost part of the cell is separated from the environment by the cell membrane. Therefore, the extraction of cell nuclei requires a suitable lysate to lyse the cell membrane. [0004] Compared with animal cells, plant cells have a c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6806C12Q1/6869
Inventor 谷晓峰冯丹
Owner THE INST OF BIOTECHNOLOGY OF THE CHINESE ACAD OF AGRI SCI
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