Chromium single cell 5 reagent kit

Naïve mice or mice immunized with 10⁵ pfu of TK⁻ HSV-2 5 weeks prior were infected
with 10⁵ pfu of WT HSV-2. One day later, single cells from vaginal tissue and spleen were isolated and surface stained
with anti-CD45 (30-F11), anti-CD3ε (145–2C11), anti-CD4 (GK1.5), anti-CD8α (53–6.7), and anti-CD19
(1D3) for vagina and anti-CD45 (30-F11), anti-CD19 (6D5 and 1D3), anti-CD38 (90), anti-GL7 (GL7), anti-MHC class II (I-A/I-E,
M5/114.15.2), and anti-IgD (11–26c.2a) for spleen. Stained cells were sorted by FACS Aria (BD Biosciences, Mountain View,
CA). CD45⁺CD3ε⁺CD4⁺, CD45⁺CD3ε⁺CD8α⁺,
CD45⁺CD3ε⁻CD19⁺ cells (vagina) and
CD45⁺CD19⁺IgD⁻IgG⁺MHCII⁺GL7⁻CD38⁺cells (spleen) were collected for further analysis. For B cell-sorted spleen samples, cell numbers were counted and 6,000 cells
were prepared. Cells from primary and secondary infected FRT samples were prepared at a cell count proportional to the immune cell
infiltration observed by flow cytometry. Single cell suspensions were loaded onto the Chromium Controller (10x Genomics) for
droplet formation. Single-cell RNA-seq libraries were prepared using the Chromium Single Cell 5’ Reagent Kit (10x Genomics)
and Single Cell V(D)J Kit according to manufacturer’s protocol. Samples were sequenced on the HiSeq4000 with 28 bp Read 1,
8 bp i7 index and 98 bp Read 2 for the gene expression library, and on the NovaSeq with 150 paired reads for BCR library.

Sorted cells were stained with Trypan blue and Countess II Automated Cell Counter (ThermoFisher) was used to assess both cell number and viability. Following QC, the single cell suspension was loaded onto Chromium Chip A (10X Genomics PN 230027) and GEM generation, cDNA synthesis, cDNA amplification, and library preparation of 10,000 cells proceeded using the Chromium Single Cell 5’ Reagent Kit (10X Genomics PN 1000006) according to the manufacturer’s protocol. cDNA amplification included 16 cycles and 21.5-50ng of the material was used to prepare sequencing libraries with 14-16 cycles of PCR. Indexed libraries were pooled equimolar and sequenced on a NextSeq 500 in a 26bp/91bp paired end run using the NextSeq 500/550 High Output Kit v2.5 (150 cycles, Illumina). An average of 256 million paired reads was generated per sample.

Droplet-based 5′-end scRNA-seq was performed by the 10x Genomics platform, and libraries were prepared by the Chromium Single Cell 5′ Reagent kit according to the manufacturer’s protocol (10x Genomics, CA, USA). The Cell Ranger Single-Cell Software Suite (10x Genomics) was used to perform barcode processing and transcript counting after alignment to the mm10 reference genome with default parameters. The Seurat R package (80 (link)) was applied to filter out low-quality cells, normalize gene expression profiles, and cluster cells. Cells expressing >10% mitochondrial gene counts or expressing less than 500 genes were discarded using the FilterCells function. Then, the NormalizeData function was applied to normalize and log transform the raw counts for each cell on the basis of its library size.