Genome-scale analysis of replication timing: from bench to bioinformatics

Area of Science:

• Genomics
• Molecular Biology
• Cell Biology

Background:

• Replication timing (RT) is cell type-specific and indicates genome organization changes during differentiation.
• Understanding RT is crucial for studying cellular processes and development.

Purpose of the Study:

• To describe a cost-effective protocol for analyzing genome-wide replication timing in mammalian cells.
• To provide a guide for analyzing resulting data sets, including normalization, smoothing, and domain segmentation.

Main Methods:

• Pulse labeling asynchronously cycling cells with 5-bromo-2-deoxyuridine (BrdU).
• Sorting cells into S-phase fractions via flow cytometry.
• Analyzing BrdU-labeled DNA using comparative genomic hybridization microarrays.

Main Results:

• The protocol enables analysis of replication timing at a biologically relevant resolution (tens to hundreds of kilobases).
• Methods for data normalization, scaling, quality control, smoothing, segmentation, and promoter assignment are presented.
• Clustering methods are discussed for relating replication timing to gene expression and epigenetic data.

Conclusions:

• This protocol offers a comprehensive approach to studying genome-wide replication timing.
• The methods facilitate the investigation of replication dynamics and their relationship with other genomic features and cellular processes.