Cells were grown to a density of 1.5E6 cells/ml and treated with 1mM hydroxyurea for 3 hours to stall replication forks and activate the intra-S-phase checkpoint. BrdU was added at 50ug/ml and cells were incubated for another 20 hours, resulting in labeling only of DNA sequences immediately adjacent to early activating origins of replication. Cells were frozen.