title: Epigenome profiling of specific plant cell types using a streamlined INTACT protocol and ChIP-seq.
abstract: Plants consist of many functionally specialized cell types, each with its own unique epigenome, transcriptome, and proteome. Characterization of these cell type-specific properties is essential to understanding cell fate specification and the responses of individual cell types to the environment. In this chapter we describe an approach to map chromatin features in specific cell types of Arabidopsis thaliana using nuclei purification from individual cell types with the INTACT method (isolation of nuclei tagged in specific cell types) followed by chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq). The INTACT system employs two transgenes to generate affinity-labeled nuclei in the cell type of interest, and these tagged nuclei can then be selectively purified from tissue homogenates. The primary transgene encodes the nuclear tagging fusion protein (NTF), which consists of a nuclear envelope-targeting domain, the green fluorescent protein, and a biotin ligase recognition peptide, while the second transgene encodes the E. coli biotin ligase (BirA), which selectively biotinylates NTF. Expression of NTF and BirA in a specific cell type thus yields nuclei that are coated with biotin and can be purified by virtue of their affinity for streptavidin-coated magnetic beads. Compared with the original INTACT nuclei purification protocol, the procedure presented here is greatly simplified and shortened. After nuclei purification, we provide detailed instructions for chromatin isolation, shearing, and immunoprecipitation. Finally, we present a low input ChIP-seq library preparation protocol based on the nano-ChIP-seq method of Adli and Bernstein, and we describe multiplex Illumina sequencing of these libraries to produce high quality, cell type-specific epigenome profiles at a relatively low cost. The procedures given here are optimized for Arabidopsis but should be easily adaptable to other plant species.
authors.affiliation: Department of Biology, O. Wayne Rollins Research Center, Emory University, 1510 Clifton Road NE, Atlanta, GA, 30322, USA.
authors.forename: Dongxue
authors.initials: D
authors.lastname: Wang
authors.forename: Roger B
authors.initials: RB
authors.lastname: Deal
mesh_terms: D002467:Cell Nucleus; D047369:Chromatin Immunoprecipitation / Q000379:methods; D044127:Epigenesis, Genetic; D057890:Epigenomics / Q000379:methods; D020869:Gene Expression Profiling / Q000379:methods; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods; D059828:Plant Cells / Q000378:metabolism*; D013347:Subcellular Fractions