title: Chromatin immunoprecipitation with fixed animal tissues and preparation for high-throughput sequencing.
abstract: Chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-Seq) is a powerful method used to identify genome-wide binding patterns of transcription factors and distribution of various histone modifications associated with different chromatin states. In most published studies, ChIP-Seq has been performed on cultured cells grown under controlled conditions, allowing generation of large amounts of material in a homogeneous biological state. Although such studies have provided great insight into the dynamic landscapes of animal genomes, they do not allow the examination of transcription factor binding and chromatin states in adult tissues, developing embryonic structures, or tumors. Such knowledge is critical to understanding the information required to create and maintain a complex biological tissue and to identify noncoding regions of the genome directly involved in tissues affected by complex diseases such as autism. Studying these tissue types with ChIP-Seq can be challenging due to the limited availability of tissues and the lack of complex biological states able to be achieved in culture. These inherent differences require alterations of standard cross-linking and chromatin extraction typically used in cell culture. Here we describe a general approach for using small amounts of animal tissue to perform ChIP-Seq directed at histone modifications and transcription factors. Tissue is homogenized before treatment with formaldehyde to ensure proper cross-linking, and a two-step nuclear isolation is performed to increase extraction of soluble chromatin. Small amounts of soluble chromatin are then used for immunoprecipitation (IP) and prepared for multiplexed high-throughput sequencing.
authors: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520.
authors: Justin L
authors: JL
authors: Cotney
authors: Department of Genetics, Yale University School of Medicine, New Haven, Connecticut 06520.
authors: James P
authors: JP
authors: Noonan
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D016133:Polymerase Chain Reaction