适用于多种禾本科植物染色质转座酶可及性测序的提核建库方法

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

为建立适用于多种禾本科植物染色质转座酶可及性测序(assay for transposase accessible chromatin with high-throughput sequencing, ATAC-seq)的细胞核提取和建库体系,本试验以水稻、小麦和结缕草的叶片及根系为研究材料,通过控制变量试验确定最佳的细胞核提取方法:采用液氮研磨样品,初步提核离心速度选取1200×g,2次ORB溶液去除细胞器等杂质,蔗糖梯度缓冲液纯化细胞核。通过构建文库、库检及测序分析发现,所提取的细胞核适用于染色质转座酶可及性测序的建库,可获得高质量测序数据。本试验建立的适用于多种禾本科植物的提核体系为获取禾本科植物染色质开放区域信息以及深入研究基因表达的调控方式奠定了基础。

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.