Morao Ana Karina, Caillieux Erwann, Colot Vincent, Roudier François
Centre National de la Recherche Scientifique (CNRS) UMR8197, Institut de Biologie de l'Ecole Normale Supérieure, Institut National de la Santé et de la Recherche Médicale (INSERM) U1024, Ecole Normale Supérieure, 46 rue d'Ulm, 75230, Paris Cedex 05, France.
Laboratoire Reproduction et Développement des Plantes, Univ Lyon, ENS de Lyon, UCB Lyon 1, CNRS, INRA, F-69342, Lyon, France.
Methods Mol Biol. 2018;1675:111-130. doi: 10.1007/978-1-4939-7318-7_8.
Progression of a cell along a differentiation path is characterized by changes in gene expression profiles. Alterations of these transcriptional programs result from cell type-specific transcription factors that act in a dynamic chromatin environment. Understanding the precise contribution of these molecular factors during the differentiation process requires accessing specific cell types within a developing organ. This chapter describes a streamlined and alternative version of INTACT, a method enabling the isolation of specific cell populations by affinity-purification of tagged nuclei and the subsequent analysis of gene expression, transcription factor binding profiles, as well as chromatin state at a genome-wide scale. In particular, modifications of the nuclei isolation, capture, and purification procedures are proposed that improve time scale, yield, and purity. In addition, the combination of different tags enables the analysis of distinct cell populations from a single transgenic line and the subtractive purification of subpopulations of cells, including those for which no specific promoter is available. Finally, we describe a chromatin immunoprecipitation protocol that has been successfully used to profile histone modifications and other chromatin-associated proteins such as RNA Polymerase II in different cell populations of the Arabidopsis root, including the quiescent center of the stem cell niche.
细胞沿着分化路径的进展以基因表达谱的变化为特征。这些转录程序的改变源于在动态染色质环境中起作用的细胞类型特异性转录因子。要了解这些分子因子在分化过程中的精确作用，需要获取发育中器官内的特定细胞类型。本章描述了INTACT的简化版替代方法，该方法能够通过对标记细胞核进行亲和纯化来分离特定细胞群体，并随后在全基因组范围内分析基因表达、转录因子结合谱以及染色质状态。特别是，提出了对细胞核分离、捕获和纯化程序的改进，以提高时间效率、产量和纯度。此外，不同标签的组合能够分析来自单个转基因系的不同细胞群体，并对细胞亚群进行减法纯化，包括那些没有可用特异性启动子的细胞亚群。最后，我们描述了一种染色质免疫沉淀方案，该方案已成功用于分析拟南芥根不同细胞群体中的组蛋白修饰和其他与染色质相关的蛋白质，如RNA聚合酶II，包括干细胞龛的静止中心。
