DNA fiber spreads were prepared as described previously (36 (link)). Cells were labeled with 25 μM IdU (5-iodo-2′-deoxyuridine), washed with warm media and then exposed to 50 μM CldU (5-Chloro-2′-deoxyuridine). Cells were lysed with the spreading buffer (200 mM Tris–HCl pH 7.5, 50 mM EDTA and 0.5% SDS) and DNA fiber were stretched onto glass slides. The DNA fibers were denatured with 2.5 M HCl for 1 h, washed with PBS and blocked with 2% BSA in PBS-Tween 20 for 60 min. IdU replication tracts were revealed with a mouse anti-BrdU/IdU antibody (BD Bioscience) and CldU tracts with a rat anti-BrdU/CldU antibody (Abcam). DNA fibers were uniformly labeled with a mouse anti-human single-stranded DNA antibody (Millipore). The secondary antibodies used for the assay were: alexa fluor 488 anti-mouse antibody (Life technologies), alexa fluor 647 anti-mouse antibody (Life technologies) and Cy3 anti-rat antibody (Jackson Immunoresearch). Replication tracts were analyzed with ImageJ software. The probability that two datasets stem from the same distribution was assayed by a non-parametrical Mann–Whitney test (Prism Software).