title: Preparation of Low-Input and Ligation-Free ChIP-seq Libraries Using Template-Switching Technology.
abstract: Chromatin immunoprecipitation (ChIP) followed by high-throughput sequencing (ChIP-seq) has become the gold standard for mapping of transcription factors and histone modifications throughout the genome. However, for ChIP experiments involving few cells or targeting low-abundance transcription factors, the small amount of DNA recovered makes ligation of adapters very challenging. In this unit, we describe a ChIP-seq workflow that can be applied to small cell numbers, including a robust single-tube and ligation-free method for preparation of sequencing libraries from sub-nanogram amounts of ChIP DNA. An example ChIP protocol is first presented, resulting in selective enrichment of DNA-binding proteins and cross-linked DNA fragments immobilized on beads via an antibody bridge. This is followed by a protocol for fast and easy cross-linking reversal and DNA recovery. Finally, we describe a fast, ligation-free library preparation protocol, featuring DNA SMART technology, resulting in samples ready for Illumina sequencing. © 2016 by John Wiley & Sons, Inc.
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Nathalie
authors.initials: N
authors.lastname: Bolduc
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Alisa P
authors.initials: AP
authors.lastname: Lehman
authors.affiliation: Takara Bio USA, Inc. (formerly Clontech Laboratories, Inc.), Mountain View, California.
authors.forename: Andrew
authors.initials: A
authors.lastname: Farmer
mesh_terms: D000818:Animals; D047369:Chromatin Immunoprecipitation / Q000379:methods*; D004247:DNA / Q000235:genetics; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D042421:Histone Code; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods; D017422:Sequence Analysis, DNA / Q000379:methods*; D014157:Transcription Factors / Q000235:genetics
chemical_list: D014157:Transcription Factors; D004247:DNA
keywords: ChIP; ChIP-seq; NGS; SMART; template switching