Nb100 142

MRE11 ChIP-seq (Paiano et al. 2020 (link)) and RPA single-strand DNA sequencing (Khil et al. 2012 (link); Tubbs et al. 2018 (link)) were performed as previously described. Twenty million pre-B cells were used per library for ChIP-seq and SSDS. Cells were fixed in 1% formaldehyde in culture media (Sigma F1635) for 10 min at 37°C, quenched with 125 mM glycine (Sigma), washed twice with cold 1× PBS, snap-frozen on dry ice, and stored at −80°C until sonication and ChIP. Sonication, immunoprecipatation, and library preparation were performed as previously detailed (Tubbs et al. 2018 (link); Paiano et al. 2020 (link)). Prior to all immunoprecipatations, sheared chromatin was precleared with 40 µL of Dynabeads Protein A (Thermo Fisher) for 30 min at 4°C. For MRE11 ChIP-seq, sheared chromatin was enriched with 4 µL of anti-MRE11 (Novus NB100-142) bound to Dynabeads Protein A overnight at 4°C. For RPA SSDS, sheared chromatin was enriched with 10 µg of anti-RPA32/RPA2 antibody (Abcam ab10359) on Dynabeads Protein A overnight at 4°C. During library preparation, kinetic enrichment of single-strand DNA was performed (Khil et al. 2012 (link)), thus sequencing only RPA-bound ssDNA, by heating sheared DNA fragments for 3 min at 95°C and allowing DNA to return to room temperature.