Cells were treated with 1mM hydroxyurea for 3 hours to stall replication forks and activate the intra-S-phase checkpoint. Cells were then split into two samples: early and late timing. To the early timing sample, BrdU was added to 50ug/ml to label sites of early replication. Both early and late samples were incubated another 21 hours in 1mM hydroxyurea, then washed with cold PBS. The early timing sample was incubated another 1hr with 50ug/ml BrdU, then frozen. The late timing sample was incubated for 4 hours after washing, then treated with 50ug/ml BrdU to label sites of late replication.