title: The INTACT method for cell type-specific gene expression and chromatin profiling in Arabidopsis thaliana.
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.
authors.affiliation: Basic Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Roger B
authors.initials: RB
authors.lastname: Deal
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
mesh_terms: D017360:Arabidopsis / Q000166:cytology / Q000235:genetics*; D001710:Biotin / Q000032:analysis; D002467:Cell Nucleus; D002843:Chromatin / Q000302:isolation & purification; D047369:Chromatin Immunoprecipitation; D020869:Gene Expression Profiling / Q000379:methods*; D018506:Gene Expression Regulation, Plant; D023281:Genomics / Q000379:methods; D030821:Plants, Genetically Modified
chemical_list: D002843:Chromatin; D001710:Biotin