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Nat Protoc. 2011 Jan;6(1):56-68. doi: 10.1038/nprot.2010.175. Epub 2010 Dec 16.

The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.

Author information

1
Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.

Abstract

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 (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 types, 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 2 d. 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.

PMID:
21212783
DOI:
10.1038/nprot.2010.175
[Indexed for MEDLINE]

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