title: Measuring genome-wide nucleosome turnover using CATCH-IT.
abstract: The dynamic interplay between DNA-binding proteins and nucleosomes underlies essential nuclear processes such as transcription, replication, and DNA repair. Manifestations of this interplay include the assembly, eviction, and replacement of nucleosomes. Hence, measurements of nucleosome turnover kinetics can lead to insights into the regulation of dynamic chromatin processes. In this chapter, we describe a genome-wide method for measuring nucleosome turnover that uses metabolic labeling followed by capture of newly synthesized histones, which we have termed Covalent Attachment of Tagged Histones to Capture and Identify Turnover (CATCH-IT). Although CATCH-IT can be used with any genome-wide mapping procedure, high-resolution profiling is attainable using paired-end sequencing of native chromatin. Our protocol also includes an efficient Solexa DNA sequencing library preparation protocol that can be used for single base-pair resolution mapping of both nucleosome and subnucleosomal particles. We not only describe the use of these protocols in the context of a Drosophila cell line but also provide the necessary changes for adaptation to other model systems.
authors.affiliation: Division of Basic Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA.
authors.forename: Sheila S
authors.initials: SS
authors.lastname: Teves
authors.forename: Roger B
authors.initials: RB
authors.lastname: Deal
authors.forename: Steven
authors.initials: S
authors.lastname: Henikoff
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D002874:Chromosome Mapping / Q000379:methods*; D003470:Culture Media / Q000378:metabolism; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D004330:Drosophila / Q000166:cytology*; D015723:Gene Library; D049750:Genome, Insect; D006657:Histones / Q000235:genetics / Q000378:metabolism; D009707:Nucleosomes / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D003470:Culture Media; D006657:Histones; D009707:Nucleosomes; D004247:DNA