5 NF-κB and AKT signaling prevent DNA damage by suppressing RAG1/2 | 145 agarose in 20 mM EDTA. Genomic DNA was carefully isolated from agarose-embedded cells by sequential overnight proteinase K treatments (1 mg/mL proteinase K, 1% SDS, 50 mM Tris pH=8, 20 mM EDTA) to prevent mechanical shearing of the DNA. After proteinase K inactivation and washing, 1/5th of each sample (200,000 cell equivalents) was used for DNA doublestranded break linker ligation. Double-stranded linkers were prepared by allowing the oligonucleotides BW-1 and BW-2 to anneal. Linkers were annealed to genomic DNA by T4 ligase in 1x ligase buffer to which 3.5% polyethyleneglycol-4000 was added. Ligation-mediated PCR (LM-PCR) was performed using a Jk2-specific forward primer (Jk2ko5) and a linker-specific primer (BW-H) in a touchdown-PCR: 95°C, 10’; 11x (94°C, 30” / 7060°C, 2.5’, with ΔT = 1°C/cycle); 29x (94°C, 30”; 60°C, 2.5’); 72°C, 10’. LM-PCR was performed on three-fold dilutions of linker-ligated genomic DNA, which was normalized for input by GAPDH PCR. PCR products were resolved in 3% agarose gels. Bands of the expected size were excised from the gel and cloned into the pCR2.1-TOPO vector for sequencing. After cloning, bacterial clones were sequenced using plasmid-specific M13 primers. Primer sequences are listed in Supplemental Table 1. Primary mouse bone marrow pre-B cell cultures Bone marrow (BM) cells were harvested from 8-20 wks old mice by flushing femurs and tibia. Total BM cells were cultured in 6-wells plates at a density of 2x106 cells/mL in RPMI1640 with 2 mM L-glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, 10% fetal calf serum, 50 mM β-mercaptoethanol, 10 ng/mL recombinant mouse IL7, and 10 ng/mL Flt3L (both from ProSpec Inc., Ness-Ziona, Israel). Culture medium and cytokines were replaced every three days. Cells were harvested after 7-10 days and analyzed by flow-cytometry, typically yielding >95% B220+ , IgM- , CD43+ progenitor B cells (data not shown). To induce large to small pre-B cell transition, cells were washed 3 times in culture medium without cytokines and placed back in 6-wells plates at 2x106 cells/mL in medium without cytokines (IL7 withdrawal). Supplementary references 1. Guikema JE, Gerstein RM, Linehan EK, et al. Apurinic/Apyrimidinic endonuclease 2 is necessary for normal B cell development and recovery of lymphoid progenitors after chemotherapeutic challenge. J Immunol. 2010;186(4):1943–1950. 2. Guikema JE, Linehan EK, Esa N, et al. Apurinic/Apyrimidinic Endonuclease 2 Regulates the Expansion of Germinal Centers by Protecting against Activation-Induced Cytidine Deaminase-Independent DNA Damage in B Cells. J. Immunol. 2014;193(2):931–9.
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