Quantitative RT-PCR of IL-7-Cultured B Cells

For qRT-PCR on IL-7-cultured B cell precursors, cells were typically harvested on day 7 after transduction, dead/apoptotic cell-depleted, and snap-frozen on dry ice. For thymus and BM, cells were isolated freshly from 53BP1^−/− mice and snap-frozen on dry ice. Stimulated B cells were obtained from the spleen and cultured as described previously (Feldhahn et al., 2012 (link)). RNA was isolated as for RNA-seq, and cDNA was generated using the RevertAid cDNA synthesis kit (Thermo Fisher Scientific). qPCR was performed using a SYBR green master mix (Sigma JumpStart) on a StepOnePlus platform (Thermo Fisher Scientific). Information of primers, PCR conditions, and analysis can be found in the Supplemental Experimental Procedures. Analysis of the IGH locus for genomic loss was done as by Jankovic et al. (2013) (link) using the primers and conditions described.