一种简单、稳健、低成本、低投入的 ChIP-seq 方法，用于分析植物中的组蛋白修饰和 Pol II。

IF 8.3 1区生物学 Q1 PLANT SCIENCES

New Phytologist Pub Date : 2024-09-15 DOI:10.1111/nph.20125

Danling Zhu, Yi Wen, Yifang Tan, Xi Chen, Zhe Wu

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引用次数: 0

摘要

染色质免疫沉淀和测序（ChIP-seq）是表观遗传和分子遗传研究的重要工具。虽然 ChIP-seq 已被常规使用，但其价格昂贵，需要数克的植物材料，而且对于富含脂肪酸的样本（如种子）来说具有挑战性。在此，我们开发了一种超灵敏植物 ChIP-seq （UP-ChIP）方法，该方法基于原生 ChIP-seq 与基于 Tn5 标记的文库构建策略相结合。UP-ChIP 一般适用于分析多种植物样本中的组蛋白修饰和 Pol II，如单一拟南芥幼苗、少量拟南芥种子和分选的细胞核。与传统的 ChIP-seq 相比，UP-ChIP 的劳动强度更低，每次 IP 只需消耗 1 μg 抗体和 10 μl 蛋白-A/G 共轭珠，而且可以有效地处理低至几毫克的起始材料。通过在不同条件和基因型下进行 UP-ChIP，我们发现 UP-ChIP 在研究组蛋白修饰方面具有高度可靠性、灵敏性和定量性。我们提供了详细的 UP-ChIP 方案。我们建议用 UP-ChIP 替代传统的 ChIP-seq 来分析组蛋白修饰和 Pol II，因为它具有降低劳动强度、减少成本和低样品输入量的优点。

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A simple, robust, cost-effective, and low-input ChIP-seq method for profiling histone modifications and Pol II in plants

Chromatin immunoprecipitation and sequencing (vs ChIP-seq) is an essential tool for epigenetic and molecular genetic studies. Although being routinely used, ChIP-seq is expensive, requires grams of plant materials, and is challenging for samples that enrich fatty acids such as seeds.
Here, we developed an Ultrasensitive Plant ChIP-seq (UP-ChIP) method based on native ChIP-seq combined with Tn5 tagmentation-based library construction strategy. UP-ChIP is generally applicable for profiling both histone modification and Pol II in a wide range of plant samples, such as a single Arabidopsis seedling, a few Arabidopsis seeds, and sorted nuclei.
Compared with conventional ChIP-seq, UP-ChIP is much less labor intensive and only consumes 1 μg of antibody and 10 μl of Protein-A/G conjugated beads for each IP and can work effectively with the amount of starting material down to a few milligrams. By performing UP-ChIP in various conditions and genotypes, we showed that UP-ChIP is highly reliable, sensitive, and quantitative for studying histone modifications. Detailed UP-ChIP protocol is provided.
We recommend UP-ChIP as an alternative to traditional ChIP-seq for profiling histone modifications and Pol II, offering the advantages of reduced labor intensity, decreased costs, and low-sample input.

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来源期刊

New Phytologist 生物-植物科学

自引率

5.30%

发文量

728

期刊介绍： New Phytologist is an international electronic journal published 24 times a year. It is owned by the New Phytologist Foundation, a non-profit-making charitable organization dedicated to promoting plant science. The journal publishes excellent, novel, rigorous, and timely research and scholarship in plant science and its applications. The articles cover topics in five sections: Physiology & Development, Environment, Interaction, Evolution, and Transformative Plant Biotechnology. These sections encompass intracellular processes, global environmental change, and encourage cross-disciplinary approaches. The journal recognizes the use of techniques from molecular and cell biology, functional genomics, modeling, and system-based approaches in plant science. Abstracting and Indexing Information for New Phytologist includes Academic Search, AgBiotech News & Information, Agroforestry Abstracts, Biochemistry & Biophysics Citation Index, Botanical Pesticides, CAB Abstracts®, Environment Index, Global Health, and Plant Breeding Abstracts, and others.