Anti rat cy3

The DNA fiber assay was performed as described previously^{44 (link)}. Briefly, asynchronous LCL, U2OS or HEK293 cells were labeled with 30 μM of CldU for 30 min, washed three times with warm PBS and then labeled with 250 μM of IdU for an additional 30 min. The reaction was terminated by treating the cells with ice-cold PBS. Cells were lysed, and DNA fibers were spread onto glass slides, fixed with methanol and acetic acid, denatured with HCl, blocked with 2% BSA and stained with anti-rat and anti-mouse 5-bromo-2′-deoxyuridine (BrdU) that specifically recognize either CldU (Sigma, C6891) or IdU (Sigma, 17125). Anti-rat Cy3 (dilution 1:300, Jackson ImmunoResearch, 712-116-153) and anti-mouse Alexa-488 (dilution 1:300, Molecular Probes, A11001) were used as the respective secondary antibodies. Microscopy was done using a Leica DMRB microscope with a DFC360FX camera. The lengths of the CldU- and IdU-labeled tracts were measured by ImageJ software. Statistical analysis was done by GraphPad Prism software using unpaired t-test. For the DNA fiber assay under genotoxic stress, the second nucleotide (IdU) was incubated in the presence of 0.1 μM APH.

After isolation and FACS sorting, 2000-8000 cells per condition were incubated 30 minutes at 37°C in serum-free StemSpan SFEM medium (STEMCELL Technologies), supplemented with 1% penicillin/streptomycin and 2% L-glutamine, to reactivate their metabolism. Subsequently 19mM 5-Chloro-2'-deoxyuridine (CldU) was added to the medium for 30 min. Medium was exchanged after short centrifugation to contain 28mM 5-iodo-2'-deoxyuridine (IdU) for 30 min.
Afterwards, DNA fiber spreads were produced as previously described (Mijic et al., 2017). In short 3μL of cell suspension was added to 7μL lysis buffer (200 mM Tris-HCl, pH 7.5, 50 mM EDTA, and 0.5% (w/v) SDS) on a glass slide. After 8 min, the slides were tilted at 15–45°, and the resulting DNA spreads were air dried, fixed in 3:1 methanol/acetic acid overnight at 4 °C. The fibers were denatured with 2.5 M HCl for 1 h, washed with PBS and blocked with 0.2% Tween 20 in 1% BSA/PBS for 40 min. Anti-BrdU antibodies recognizing CldU (1:500, ab6326; Abcam) and IdU (1:100, B44, 347580; BD Biosciences) were incubated for 2.5 h in the dark at RT, followed by 1 h incubation with secondary antibodies at RT in the dark: anti–mouse Alexa Fluor 488 (1:300, Invitrogen) and anti–rat Cy3 (1:150, Jackson ImmunoResearch Laboratories, Inc.). Fibers were visualized using a Leica DMI6000 microscope (64x, oil) and analyzed using ImageJ software.