Native Chromatin Preparation and Illumina/Solexa Library Construction

Abstract

INTRODUCTIONHigh-throughput whole-genome analysis has become a practical and important technique to understand nuclear processes, such as transcription, replication, and genome structure. Though microarrays have been the preferred genome-scale analysis method for over a decade, new technologies, referred to as next-generation sequencing, offer distinct advantages over microarrays in both sensitivity and scale. Several next-generation sequencing platforms are currently available, including the Genome Analyzer (Solexa/Illumina), 454 (Roche), and ABI-SOLiD (Applied Biosystems). This protocol describes sample preparation for sequencing of chromatin-immunoprecipitated DNA (ChIP-Seq) to analyze histone modification patterns using native chromatin and the Genome Analyzer. One advantage of using native chromatin as compared to cross-linked chromatin is that it provides single-nucleosome-level resolution and avoids nonspecific modification signals from different nucleosomes carried over through protein-protein interactions. The protocol includes purification of human CD4+ T cells from lymphocytes and chromatin fragmentation using micrococcal nuclease (MNase) digestion, followed by chromatin immunoprecipitation (ChIP) and construction of a library for sequencing.