The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.
The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.
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DOI：
10.1038/nprot.2010.175
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发表时间：
2011-01
期刊：
影响因子：
14.8
通讯作者：
Henikoff S
中科院分区：
文献类型：
--
作者：
Deal RB;Henikoff S
对整个生物体内细胞分化和功能的基因组研究取决于从组织中分离特定细胞类型的能力，但这在技术上是困难的。我们开发了一种称为INTACT（用于分离特定细胞类型中标记的细胞核）的方法，该方法允许基于亲和力从组织的单个细胞类型中分离细胞核，从而避免了与机械纯化技术相关的问题。在该方法中，通过在感兴趣的细胞类型中转基因表达生物素化的核膜蛋白来亲和标记细胞核。从转基因植物中分离总细胞核，然后使用链霉亲和素包被的磁珠纯化生物素标记的细胞核，而不需要专门的设备。INTACT从所需细胞类型中获得高产量和高纯度的细胞核，可用于基因表达和染色质特征的全基因组分析。从细胞核纯化到cDNA制备或染色质免疫沉淀（ChIP）的整个过程可以在两天内完成。我们提出的方案假设转基因株系已经可用，并且包括在微阵列或深度测序分析之前扩增cDNA或ChIP DNA的程序细节。
Genomic studies of cell differentiation and function within a whole organism depend on the ability to isolate specific cell types from a tissue, but this is technically difficult. We developed a method called INTACT (for isolation of nuclei tagged in specific cell types) that allows affinity-based isolation of nuclei from individual cell types of a tissue, thereby circumventing the problems associated with mechanical purification techniques. In this method nuclei are affinity labeled through transgenic expression of a biotinylated nuclear envelope protein in the cell type of interest. Total nuclei are isolated from transgenic plants and biotin-labeled nuclei are then purified using streptavidin-coated magnetic beads without the need for specialized equipment. INTACT gives high yield and purity of nuclei from the desired cell type, which can be used for genome-wide analysis of gene expression and chromatin features. The entire procedure, from nuclei purification through cDNA preparation or chromatin immunoprecipitation (ChIP), can be completed within two days. The protocol we present assumes that transgenic lines are already available, and includes procedural details for amplification of cDNA or ChIP DNA prior to microarray or deep sequencing analysis.
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