Day14 B cells LCMV IFNAR1+/+ male spleen (GEX library); Mus musculus; RNA-Seq
Three groups of mice were used: (1) LCMV-infected IFNAR1flox/flox mice (B cells with intact IFN-I signaling), (2) LCMV-infected MB1cre IFNAR1flox/flox mice (B cell-specific deletion of IFNAR1), and (3) VSV-infected IFNAR1flox/flox mice (acute infection control). All animals were infected with either LCMV Cl13 or VSV and subsequently immunized with NP-CGG. After spleen collection, mechanical dissociation, and cell counting, B cells were enriched by depleting non-B cells using the EasySep Mouse Biotin Positive Selection Kit II (Stemcell Canada) with the following biotinylated antibodies: anti-CD4, anti-CD8, anti-TER119, anti-CD49b, anti-CD11c, anti-CD11b, and anti-Ly6G. We then followed the Cell Surface Protein Labeling for Single Cell RNA Sequencing protocol (10X Genomics) using the following TotalSeq C barcoded antibodies (BioLegend USA): C0301 (ACCCACCAGTAAGAC), C0302 (GGTCGAGAGCATTCA), C0303 (CTTGCCGCATGTCAT), and C0304 (AAAGCATTCTTCACG). Four mice per group were individually labeled with these barcoded antibodies. Within each condition, samples labeled with C0301 and C0302 were pooled together, and samples labeled with C0303 and C0304 were pooled together, resulting in two tubes per condition. Approximately 10,000 enriched B cells per sample were processed for scRNA-seq using the Chromium Next GEM Single Cell 5 Reagent Kit v2 (10X Genomics) (CG000330RevF) according to the manufacturer's recommendations. Libraries were prepared for 5' gene expression, BCR VDJ repertoire sequencing, and cell surface protein detection. cDNA and library quality were verified using the High Sensitivity DNA Kit and a 2100 BioAnalyzer (Agilent Technologies). Sequencing was performed on an Illumina NovaSeq using a paired-end dual-index strategy with the following cycle distribution: Read 1: 26 cycles, i7 index: 10 cycles, i5 index: 10 cycles, Read 2: 90 cycles. Each sample contained approximately 10,000 cells, with sequencing depth allocated as follows: BCR VDJ: 5,000 reads per cell; 5' gene expression: 20,000 reads per cell; surface protein labeling: 5,000 reads per cell.