Figure 3. Replication timing analysis by retroactive fluorescence-activated cell sorter synchronization.

This method can be applied to any proliferating cell population that can be dissociated into a single-cell suspension. Cells are stained with any dye that fluoresces proportionally to DNA content to produce a histogram of the number of cells with unreplicated (G1 phase), fully replicated (G2/M phase) or increasing (S phase) DNA content. In the 5-bromodeoxyuridine immunoprecipitation (BrdU-IP) method, cells are first pulse-labelled with BrdU to tag replicating DNA in a short (10–20%) interval of S phase and then separated into early- and late-S-phase populations by DNA content using the fluorescence-activated cell sorter (FACS). BrdU-substituted nascent DNA from these populations is immunoprecipitated, differentially labelled and co-hybridized to a high-density whole-genome oligonucleotide microarray. Alternatively, the BrdU-IP DNA can be sequenced. In the S/G1 method, unlabelled cells are sorted into either G1 phase populations (in which the copy numbers of all genomic sequences are equivalent) or total S phase populations (in which the copy numbers are proportional to how early the sequence replicates). DNA is isolated and replication timing is determined as the copy number ratio of S/G1 across the genome either on an array or by sequencing. A link to protocols for these methods, and more information, is provided in the ‘Further Information’ section.