Chromatin immunoprecipitation (ChIP) is the gold-standard method for detection of interactions between proteins and chromatin and is a powerful tool for identification of epigenetic modifications. Although ChIP protocols for plant species have been developed, many specific features of plants, especially woody plants, still hinder the efficiency of immunoprecipitation, resulting in inefficient ChIP enrichment and an active demand for a highly efficient ChIP protocol. In this study, using birch (Betula platyphylla) and Arabidopsis (Arabidopsis thaliana) as the research materials, we identified five factors closely associated with ChIP efficiency, including crosslinking, concentration of chromatin using centrifugal filters, use of a different immunoprecipitation buffer, rescue of DNA with proteinase K, and use of Suc to increase immunoprecipitation efficiency. Optimization of any these factors can significantly improve ChIP efficiency. Considering these factors together, we developed a robust ChIP protocol that achieved a 14-fold improvement in ChIP enrichment for birch and a >6-fold improvement for Arabidopsis compared to the standard ChIP method. As this ChIP method works well in both birch and Arabidopsis, it should also be suitable for other woody and herbaceous species. In addition, this ChIP method enables detection of low-abundance transcription factor-DNA interactions and may extend the application of ChIP in the plant kingdom.

染色质免疫沉淀（ChIP）是检测蛋白质与染色质之间相互作用的金标准方法，是鉴定表观遗传修饰的强大工具。尽管已经开发了针对植物物种的ChIP方案，但是植物（尤其是木本植物）的许多特定功能仍然阻碍了免疫沉淀的效率，导致ChIP富集效率低下，并且对高效ChIP方案的积极需求。在本研究中，使用桦木（Betula platyphylla）和拟南芥（Arabidopsis thaliana））作为研究材料，我们确定了与ChIP效率密切相关的五个因素，包括交联，使用离心滤器的染色质浓度，使用不同的免疫沉淀缓冲液，使用蛋白酶K拯救DNA以及使用Suc提高免疫沉淀效率。优化这些因素可以显着提高ChIP效率。综合考虑这些因素，我们开发了可靠的ChIP方案，与标准ChIP方法相比，桦木的ChIP富集度提高了14倍，拟南芥的ChIP富集度提高了6倍以上。由于此ChIP方法在桦木和拟南芥中均能很好地发挥作用，因此它也应适用于其他木本和草本物种。此外，