GSM7108359: LCMV-WE, replicate 2 + LCMV-Docile, replicate 2, VDJ; Mus musculus; OTHER
C57Bl6 mice were infected with LCMV-WE or LCMV-Docile. 28 days after infection, spleens were surgically removed from culled mice and non-RBC splenocytes were isolated. LCMV-specific B cells were magnetically enriched using an LCMV-NP tetramer system. Enriched cells were then stained with viability dye (FVS) and relevant antibodies, and 100,000 LCMV-specific B cells per sample were sorted using the BD Influx (BD Biosciences) cell sorter. After FACS, cells were spun down and resuspended in washing buffer (PBS 0.04% BSA) (50μl per ~1-2 million cells) and incubated with mouse Fc blockers (FcX, Biolegend) for 10 minutes at 4°C to block non-specific antibody binding. Following Fc blocking, each sample was incubated with either TotalSeq anti-mouse Hashtag Antibody 1 (Biolegend #155861) or 2 (Biolegend #155863) for 20 minutes at 4°C. Acute samples were tagged with Hashtag Antibody 1 and chronic samples were tagged with Hashtag Antibody 2. Following incubation, cells were washed 3x with 1ml of washing buffer to remove unbound antibodies. LCMV-WE and LCMV-Docile samples were then pooled at equal ratios in (PBS 0.04% BSA 0.2 U/ml RNAse inhibitor) at 1200 cells/ul. Cell viability >95% was confirmed via Trypan Blue staining. Reverse transcription, cDNA amplification and library preparation for single cell gene expression and V(D)J clonotypes were performed based on the manufacturers protocol using the Chromium Single Cell 5′ Library & Gel Bead Kit v3 (10X Genomics). Libraries were pooled at equimolar ratios and gene expression libraries were sequenced on the Illumina NovaSeq 6000 System, while HTO (hashtag oligo) and V(D)J (BCR) libraries were sequenced on Illumina NextSeq 500.