Fcas aria 2

For gene deletion, a pair of single guide RNAs (sgRNAs) were designed with the CRISPOR program. One plasmid expressing both gRNA, Cas9 and green fluorescent protein (LentiCRISPRV2GFP, Plasmid # 82416 Addgene) was nucleofected into Ramos cells using Amaxa (Lozano) according to the manufacturer’s protocol. At 24 h after transfection, GFP cells were sorted with BD FCAS Aria II and plated into single clones in 96-well plates. Individual clones were genotyped by PCR to identify mutated clones by insertion or deletion. Candidate clones were further confirmed by Sanger sequencing and western blot. Guide RNA sequences: pol eta gRNAfor: 5’-GGTGAGGTTAGCTTTCCCAC-3’ and pol eta gRNARev: 5’-GTGGGAAAGCTAACCT-CACC-3’, AICDA gRNAfor: 5’-GTGGAATTGCTCTTCCTCC-3’, AICDA gRNARev: 5’-GGAGGAAGAG CAATTCCAC-3’. A vector expressing full-length human polη was described previously (27 (link)), and a vector expressing full-length human AICDA was described in (21 (link)) and used for complementation of the KO cell lines. polη Zeocin- and AID puromycin-resistant clones were selected with 150 μg/mL Zeocin (Roche, Mannheim, Germany) and 600 ng/mL puromycin (Invitrogen), respectively.

B220⁺PNA^hi GC B cells were sorted with BD FCAS Aria II from Peyer’s Patches or spleens of SRBC-immunized mice. J_H4 and Jκ5 introns were PCR amplified with the indicated primers and the ~1.2 kb J_H4 fragment^{52 (link)} and ~0.8 kb Jκ5 fragment^{53 (link)} were gel-purified. The PCR products were further tagged with illumine P5 and P7 index primers and subjected to illumina HiSeq. Data were analyzed as similar as performed with S region mutations.