GSM8149339: rAd_CSP; Mus musculus; OTHER
Lymphocytes were isolated from lung tissue. First, lungs were cut into small pieces and enzymatically digested in collagenase D (250 U/ml) and DNase I (80 U/ml) diluted in 2 ml R10 medium (RPMI 1640 supplemented with 10 % FCS, 2 mM L-Glutamine, 10 mM HEPES, 50 µM β-mercaptoethanol and 1 % penicillin/streptomycin) for 45 min at 37 °C. Disaggregated lung tissues were passed through 70-μm cell strainers and red blood cells were lysed by resuspension in ammonium-chloride-potassium. Isolated lymphocytes were directly stained in PBS containing 0.5 % BSA and 1mM NaN3. Library was performed to the manufacter's instruction (Chromium Next GEM Chip K Single Cell Kit, Chromium Next GEM Single-Cell 5′ Kit v2 Kit, Dual Index Kit TT Set A, 5' Feature Barcode Kit, Chromium Single-Cell Mouse TCR Amplification Kit (all 10x Genomics, CA, USA)). Briefly, for each group, 2x105 barcoded NP147-155-specific CD8+ T cells were sorted. Samples were centrifuged, resuspended, and a master-mix containing RT Reagent B, Poly-dt RT Primer, and RT Enzyme C was added (Chromium Next GEM Single Cell 5' Reagent Kits v2, 10x Genomics). This master-mix and cell suspension mixture was loaded into Chromium Chip K (10x Genomics) and partitioned into Gel Beads In-Emulsions (GEMs) in a chromium controller (10x Genomics). ScRNA libraries as well as those for scTCR-sequencing were prepared according to the Chromium Next GEM Chip K Single Cell Kit, the Chromium Next GEM Single-Cell 5′ Kit v2 Kit, the Dual Index Kit TT Set A, the 5' Feature Barcode Kit, and the Chromium Single-Cell Mouse TCR Amplification Kit (all 10x Genomics, CA, USA). The single-cell RNA-seq (scRNA-seq) and VDJ libraries were sequenced by Novogene UK.