Quantifying DNA Double-Strand Breaks in B Cells

DNA DSB ends were labelled with biotin41 (link). Rad52^+/+ and Rad52^−/− B cells were stimulated with LPS or LPS plus IL-4 for 60 h. Live B cells were separated through a Ficoll gradient, followed by fixation, permeabilization and in situ DNA end-labelling with bio-dUTP using TdT (Fig. 6d). Chromatin was pulled down with rabbit anti-Rad52 antibody (H-300, Santa Cruz Biotechnology, 5 μg ml⁻¹), anti-Ku70/Ku86 monoclonal antibody (MA1-21818, Thermo Fisher Scientific, 5 μg ml⁻¹) or control rabbit or mouse IgG with irrelevant specificity. Genomic DNA was prepared using a QIAquick PCR purification kit (Qiagen). Biotin-labelled DNA fragments were captured by streptavidin magnetic beads (Promega) before being analysed by quantitative PCR.