Genome-wide analysis of replication timing in mammalian cells: troubleshooting problems encountered when comparing...

DNA is replicated in a defined temporal order that is developmentally regulated and constitutes a unique and stable fingerprint of a given cell type. Recently, we developed a robust assay to profile replication timing genome wide that can be applied to essentially any proliferating cell population. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU). The cells are sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated (BrdU IP), amplified, differentially labeled and co-hybridized to a whole-genome comparative genomic hybridization microarray (or sequenced). Since the basic steps of this protocol have been detailed elsewhere, here we focus on problems encountered when adapting this protocol to different cell types or tissue sources and modifications that have been successfully applied to troubleshoot these problems. There is an increasing demand for such studies to address how replication is regulated during development, its relationship to chromatin architecture and other chromosome functions, and the relevance of cell culture models to regulation in the native organismal niche.