Background Covalent modifications of histones and histone variants have great influence on chromatin structure, which is involved in the transcriptional regulation of gene expression. Chromatin immunoprecipitation (ChIP) is a powerful tool for studying in vivo DNA-histone interactions. Strawberry is a model for Rosaceae and non-climacteric fruits, in which histone modifications have been implicated to affect fruit development and ripening. However, a validated ChIP method has not been reported in strawberry, probably due to its high levels of polysaccharides which affect the quality of prepared chromatin and the efficiency of immunoprecipitation. Results We describe a native chromatin immunoprecipitation (N-ChIP) protocol suitable for strawberry by optimizing the parameters for nuclei isolation, chromatin extraction, DNA fragmentation and validation analysis using quantitative real-time PCR (qRT-PCR). The qRT-PCR results show that both the active mark H3K36me3 and the silent mark H3K9me2 are efficiently immunoprecipitated for the enriched regions. Compared to X-ChIP (cross-linked chromatin followed by immunoprecipitation), our optimized N-ChIP procedure has a higher signal-to-noise ratio and a lower background for both the active and the silent histone modifications. Furthermore, high-throughput sequencing following N-ChIP demonstrates that nearly 90% of the enriched H3K9/K14ac peaks are overlapped between biological replicates, indicating its remarkable consistency and reproducibility. Conclusions An N-ChIP method suitable for the fleshy fruit tissues of woodland strawberry Fragaria vesca is described in this study. The efficiency and reproducibility of our optimized N-ChIP protocol are validated by both qRT-PCR and high-throughput sequencing. We conclude that N-ChIP is a more suitable method for strawberry fruit tissues relative to X-ChIP, which could be combined with high-throughput sequencing to investigate the impact of histone modifications in strawberry and potentially in other fruits with high content of polysaccharides.

中文翻译：

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用于研究草莓果实中组蛋白修饰的天然染色质免疫沉淀 (ChIP) 协议。

背景 组蛋白和组蛋白变体的共价修饰对染色质结构有很大影响，染色质结构参与基因表达的转录调控。染色质免疫沉淀 (ChIP) 是研究体内 DNA-组蛋白相互作用的有力工具。草莓是蔷薇科和非更年期果实的模型，其中组蛋白修饰与影响果实发育和成熟有关。然而，尚未在草莓中报道经过验证的 ChIP 方法，这可能是由于其高水平的多糖会影响制备的染色质的质量和免疫沉淀的效率。结果 我们通过优化核分离、染色质提取、使用定量实时 PCR (qRT-PCR) 进行 DNA 片段化和验证分析。qRT-PCR 结果表明，对于富集区域，活性标记 H3K36me3 和沉默标记 H3K9me2 均被有效免疫沉淀。与 X-ChIP（交联染色质后免疫沉淀）相比，我们优化的 N-ChIP 程序具有更高的信噪比和更低的活性组蛋白修饰和沉默组蛋白修饰的背景。此外，N-ChIP 后的高通量测序表明，近 90% 的富集 H3K9/K14ac 峰在生物复制之间重叠，表明其显着的一致性和可重复性。结论 本研究描述了一种适用于林地草莓草莓肉质果实组织的 N-ChIP 方法。我们优化的 N-ChIP 协议的效率和可重复性通过 qRT-PCR 和高通量测序得到验证。我们得出结论，相对于 X-ChIP，N-ChIP 是一种更适合草莓果实组织的方法，它可以与高通量测序相结合，研究组蛋白修饰对草莓以及其他多糖含量高的水果的影响。