Abstract：
In plants, gene expression is orchestrated by thousands of transcription factors (TFs). For instance, a large set of bHLH TFs are involved in the regulation of iron homeostasis in Arabidopsis thaliana. The identification of the direct target genes of TFs through uncovering the interaction between the TFs and cis-regulatory elements has become an essential step toward a comprehensive understanding of the iron homeostasis transcriptional regulatory network in Arabidopsis. Chromatin immunoprecipitation (ChIP) followed by qRT-PCR (ChIP-qPCR), sequencing (ChIP-seq), or chip hybridization (ChIP-chip) is a robust tool to investigate protein-DNA interactions in plants in a physiological context. The procedure generally includes six steps: DNA-protein crosslink, isolation of nuclei, shearing of chromatin, immunoprecipitation, DNA purification, and qRT-PCR analyses. In this protocol, we describe guidelines, experimental setup, and conditions for ChIP experiment in Arabidopsis. This protocol focuses on seedlings grown in control and iron deficiency conditions, but can readily be adapted for use with other Arabidopsis tissues or samples. In addition, the protocol could also be applied to perform ChIP-chip or ChIP-seq experiments.
摘要：
在植物中,基因表达由成千上万的转录因子(TFs)协调。例如,大量的bHLHTF参与拟南芥铁稳态的调节。通过揭示TFs与顺式调控元件之间的相互作用来鉴定TFs的直接靶基因已成为全面了解拟南芥铁稳态转录调控网络的重要步骤。染色质免疫沉淀（ChIP），然后qRT-PCR（ChIP-qPCR），测序(ChIP-seq)，或芯片杂交（ChIP-chip）是在生理背景下研究植物中蛋白质-DNA相互作用的强大工具。该程序通常包括六个步骤：DNA-蛋白质交联，原子核的分离，染色质的剪切，免疫沉淀,DNA纯化,和qRT-PCR分析。在这个协议中,我们描述指导方针，实验装置，拟南芥ChIP实验的条件。该协议侧重于在控制和缺铁条件下生长的幼苗，但可以很容易地适用于其他拟南芥组织或样品。此外,该协议也可以应用于执行ChIP芯片或ChIP-seq实验。
