利用靶向切割和标签化技术对植物中的 H3K4me3 修饰进行分析。

Profiling of H3K4me3 Modification in Plants using Cleavage under Targets and Tagmentation.

机构信息

College of Agriculture and Biotechnology, Zhejiang University; Central Laboratory, State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Zhejiang Academy of Agricultural Sciences.

State Key Laboratory of Crop Genetics and Germplasm Enhancement, Cotton Hybrid R & D Engineering Center (the Ministry of Education), College of Agriculture, Nanjing Agricultural University.

出版信息

J Vis Exp. 2022 Apr 22(182). doi: 10.3791/62534.

DOI:10.3791/62534

PMID:35532268

Abstract

Epigenomic regulation at the chromatic level, including DNA and histone modifications, behaviors of transcription factors, and non-coding RNAs with their recruited proteins, lead to temporal and spatial control of gene expression. Cleavage under targets and tagmentation (CUT&Tag) is an enzyme-tethering method in which the specific chromatin protein is firstly recognized by its specific antibody, and then the antibody tethers a protein A-transposase (pA-Tn5) fusion protein, which cleaves the targeted chromatin in situ by the activation of magnesium ions. Here, we provide our previously published CUT&Tag protocol using intact nuclei isolated from allortetraploid cotton leaves with modification. This step-by-step protocol can be used for epigenomic research in plants. In addition, substantial modifications for plant nuclei isolation are provided with critical comments.

摘要

染色质水平的表观遗传调控，包括 DNA 和组蛋白修饰、转录因子的行为以及与其募集的蛋白质一起的非编码 RNA，导致基因表达的时空控制。靶向切割和标签化（CUT&Tag）是一种酶连接方法，其中特定的染色质蛋白首先被其特异性抗体识别，然后抗体连接一个蛋白 A-转座酶（pA-Tn5）融合蛋白，通过镁离子的激活，在原位切割靶向染色质。在这里，我们提供了之前发表的使用经修饰的异源四倍体棉花叶片分离的完整核进行 CUT&Tag 的协议。本分步协议可用于植物的表观基因组学研究。此外，还提供了用于植物核分离的大量修改，并附有重要评论。