GSM7900922: WT mouse germinal-center B cells BCR-seq rep2; Mus musculus; OTHER
Splenic naive and germinal-center (GC) B cells were enriched using a B cell isolation kit followed by FACS-sorting. Mice were immunized with NP-OVA and leukocytes were harvested from spleen by passage through a 40-μm mesh and incubated for 10 min with 1 μg/mL of anti-CD16/32 Fc-block in FACS buffer (PBS supplemented with 1 mM EDTA, 2% heat-inactivated FBS, and 0.02% sodium azide). Cells were further labeled using fluorochrome-coupled antibodies directed against the respective antigens: CD45R, GL-7, CD95, CD38 and CD138.  Cells were surface-stained at 4°C for 30 minutes, and subsequently, GC B cells (CD45R+, GL7+, CD95+) and naïve B cells (IgD+, CD45R+, GL7-, CD38-) were sorted either using a FACS Aria II (Becton Dickinson) or SH-800 cell sorter (Sony). Libraries for sequencing were prepared as described (17). Briefly, genomic DNA was sonicated and subjected to linear amplification-mediated PCR using biotinylated bait primers. Linear amplification-mediated PCR products were purified using Dynabeads MyOne streptavidin C1 magnetic beads (Thermo Fisher Scientific, MA, USA) and ligated to bridge adaptors. Adaptor-ligated products were amplified by nested PCR with indexed JH1/JH3 primers as well as primers annealed to the adaptor. PCR products were further tagged with Illumina sequencing adaptor sequences and size-selected via gel extraction. Libraries were sequenced by paired-end 300-bp sequencing on MiSeq, an Illumina sequencing platform (San Diego, CA, USA). Rep-SHM-Seq was performed as described previously using our established bioinformatics pipeline (18). We used bait primers close to the coding ends of JHs to capture full-length V(D)J sequences for SHM analysis and used Illumina MiSeq 2 × 300-bp paired-end sequencing (19, 53). As described before, mixed JH primers were designed based on a highly degenerate region to minimize the amplification bias caused by different primers. 400 ng genomic DNA from FACS-sorted naive or GC B cells was used as starting material to keep the level of PCR amplification comparable among different samples. In addition, to minimize the potential cross-contamination due to FACS sorting, we further filtered reads by assigning mutated sequences to GC B cell samples and non-mutated sequences to naive B cell samples.