GSM8488674: B,LCMV,TCR,S4; Mus musculus; RNA-Seq
For the RRV and LCMV samples, cells from the popliteal draining lymph node of infected mice were pooled. For the naive sample, cells were pooled from uninfected wild type and P14 transgenic mouse draining lymph nodes. A portion of each sample was enriched for transgenic CD8+ T cells by magnetic bead negative selection and fluorescence-activated cell sorted for the CD45.2 congenic marker and added to the bulk sample at a 1:4 ratio. Approximately 20,000 cells per sample were subjected to gel bead-in-emulsion (GEM) generation, reverse transcription, and construction of libraries for sequencing according to the protocol provided in the Chromium Single Cell 5' Reagent Kit v2 with TCR enrichment manual (10X Genomics). Briefly, cDNA was prepared after GEM generation and barcoding, followed by the GEM-RT reaction and bead cleanup steps. Purified cDNA was amplified for 11-13 cycles before clean-up using SPRI select beads (Beckman Coulter, B23318). Samples were then run on a Bioanalyzer to determine cDNA concentrations. Gene expression libraries (GEX libraries) were prepared as recommended by the 10x Genomics Chromium Single Cell 5' Reagent Kit v2 with TCR enrichment user guide with appropriate modifications to the PCR cycles based on the calculated cDNA concentration. For sample preparation on the 10x Genomics platform, the Chromium Single Cell 5' Reagent Kit v2 with TCR enrichment, Chromium Next GEM Chip G Single Cell Kit, and Dual Index Kit TT Set A were used.  The concentration of each library was determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer's protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument. Normalized libraries were sequenced on a NovaSeq6000 S4 Flow Cell using the XP workflow according to manufacturer protocol. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.