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

Publication Type:

Journal Article

Source:

Nature protocols, Volume 6, Issue 1, p.56-68 (2011)

Keywords:

2011, Arabidopsis, Basic Sciences Division, Biotin, Cell Nucleus, Center-Authored Paper, chromatin, Chromatin Immunoprecipitation, Gene Expression Profiling, Gene Expression Regulation, Plant, GENOMICS, Genomics Core Facility, Plants, Genetically Modified, Scientific Imaging Core Facility, Shared Resources

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.