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Acta Agronomica Sinica ›› 2023, Vol. 49 ›› Issue (7): 1799-1807.doi: 10.3724/SP.J.1006.2023.22041

• CROP GENETICS & BREEDING·GERMPLASM RESOURCES·MOLECULAR GENETICS • Previous Articles     Next Articles

A method of nuclei extraction and library construction for chromatin transposase accessibility sequencing in gramineous plants

QI Ze-Wen1,2(), HUANG Ming-Han2, ZHANG Jia-Hui2, LIU Yi2, HAN Lie-Bao1,*(), HE Hang2,*()   

  1. 1College of Grassland Science, Beijing Forestry University, Beijing 10083, China
    2School of Advanced Agricultural Sciences, Peking University, Beijing 10081, China
  • Received:2022-07-01 Accepted:2022-11-25 Online:2023-07-12 Published:2022-12-01
  • Contact: *E-mail: hanliebao@163.com; E-mail: hehang@pku.edu.cn E-mail:qizewen0404@163.com;hanliebao@163.com;hehang@pku.edu.cn
  • Supported by:
    The National Natural Science Foundation of China(31971770)

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Abstract:

In order to establish a system for extracting nuclei and constructing library of assay for transposase accessible chromatin with high-throughput sequencing (ATAC-seq) in gramineous plants, the leaves and roots of rice, wheat, and zoysia were used as the research materials. The best method to extract nuclei was determined by designing control variable experiments: grinding samples with liquid nitrogen, selecting 1200×g centrifugal stress for extracting initial nuclei, using ORB buffer twice and SCB buffer to purify nuclei. It was suggested that the extracted nuclei were suitable for the sequencing of transposase accessible chromatin and high-quality sequencing data could be obtained by testing. In summary, an integrated system suitable for gramineous plants to extract nuclei had been established. This method will lay a foundation for obtaining the information of chromatin accessible regions and the regulation of gene expression in gramineous plants.

Key words: gramineae, chromatin accessibility, ATAC-seq, nuclei

Fig. 1

Nuclei under optical microscope A: the nuclei extracted by using the leaves of fresh rice (Bright blue dots are nuclei); B: the nuclei extracted by using the leaves of rice with liquid nitrogen (Bright blue dots are nuclei)."

Fig. 2

Number of the nuclei extracted under different centrifugal forces A: the nuclei extracted by using rice tissues; B: the nuclei extracted by using wheat tissues; C: the nuclei extracted by using zoysia tissues."

Fig. 3

Nucleus extracted by different purification methods A: the nuclei purified with 20 μL ORB buffer; B: the nuclei purified with 10 mL ORB buffer; C: the nuclei purified with ORB buffer twice; D: the detection of broken nuclei with trypan blue."

Fig. 4

Numbers and the distribution patterns of chromatin accessible regions A: the distribution patterns of chromatin accessible regions in wheat; B: the distribution patterns of chromatin accessible regions in rice; C: the distribution patterns of chromatin accessible regions in zoysia; D: Wayne diagram of chromatin accessible regions in wheat; E: Wayne diagram of chromatin accessible regions in rice; F: the number of chromatin accessible regions."

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