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

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

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Arabidopsis / cytology
  • Arabidopsis / genetics*
  • Biotin / analysis
  • Cell Nucleus
  • Chromatin / isolation & purification
  • Chromatin Immunoprecipitation
  • Gene Expression Profiling / methods*
  • Gene Expression Regulation, Plant
  • Genomics / methods
  • Plants, Genetically Modified

Substances

  • Chromatin
  • Biotin