title: Chromatin immunoprecipitation (ChIP) of plant transcription factors followed by sequencing (ChIP-SEQ) or hybridization to whole genome arrays (ChIP-CHIP).
abstract: Chromatin immunoprecipitation (ChIP) is a powerful technique to study interactions between transcription factors (TFs) and DNA in vivo. For genome-wide de novo discovery of TF-binding sites, the DNA that is obtained in ChIP experiments needs to be processed for sequence identification. The sequences can be identified by direct sequencing (ChIP-SEQ) or hybridization to microarrays (ChIP-CHIP). Given the small amounts of DNA that are usually obtained in ChIP experiments, successful and reproducible sample processing is challenging. Here we provide a detailed procedure for ChIP of plant TFs, as well as protocols for sample preparation for ChIP-SEQ and for ChIP-CHIP. Our ChIP procedure is optimized for high signal-to-noise ratio starting with tissue fixation, followed by nuclei isolation, immunoprecipitation, DNA amplification and purification. We also provide a guide for primary data analysis of ChIP-SEQ data. The complete protocol for ChIP-SEQ/ChIP-CHIP sample preparation starting from plant harvest takes approximately 7 d.
authors.affiliation: Laboratory of Molecular Biology, Wageningen University, Wageningen, The Netherlands.
authors.forename: Kerstin
authors.initials: K
authors.lastname: Kaufmann
authors.forename: Jose M
authors.initials: JM
authors.lastname: Muiño
authors.forename: Magne
authors.initials: M
authors.lastname: Østerås
authors.forename: Laurent
authors.initials: L
authors.lastname: Farinelli
authors.forename: Pawel
authors.initials: P
authors.lastname: Krajewski
authors.forename: Gerco C
authors.initials: GC
authors.lastname: Angenent
mesh_terms: D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism; D029681:Arabidopsis Proteins / Q000302:isolation & purification / Q000378:metabolism; D001665:Binding Sites / Q000235:genetics; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D018744:DNA, Plant / Q000235:genetics* / Q000378:metabolism*; D018745:Genome, Plant; D017403:In Situ Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods; D010940:Plant Proteins / Q000302:isolation & purification* / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000302:isolation & purification* / Q000378:metabolism*
chemical_list: D029681:Arabidopsis Proteins; D018744:DNA, Plant; D010940:Plant Proteins; D014157:Transcription Factors