GSM5904619
Lymphocytes from the lung were harvested 0, 3, 5, 7, 10, and 21 days post infection. Lungs lymphocytes were isolated into sort buffer (1XPBS/2% FBS/2mM EDTA) and processed as stated previously.  Single-cell suspensions were stained with 2.5ug of anti-CD4-APC (RM4-5, Biolegend 100516) antibody in 1mL of sort buffer and incubated at 4°C for 30 minutes.  Following wash, cells were resuspended in 50ul of anti-APC microbeads (Miltenyi) and 350ul of sort buffer.  Cells were then washed and run over an LS column (Miltenyi).  The bound fraction was collected and stained with ghost dye red 780 viability dye (Tonbo Biosciences 13-0865-T100), antibodies against TCRbeta (H57-597, Biolegend 109229), CD8alpha (53-6.7, ThermoFisher Scientific 47-0081-82), Gr1 (RB6-8C5, ThermoFisher Scientific 47-5931-82), NK1.1 (PK136, ThermoFisher Scientific 47-5941-82), CD11b (M1/70, ThermoFisher Scientific 47-0112-82), CD11c (N418, ThermoFisher Scientific 47-0114-82), F4/80 (BM8, ThermoFisher Scientific 47-4801-82), and Ter119 (ThermoFisher Scientific 47-5921-82), 0.25ug total-seq C streptavidin-PE (C0951, Biolegend 405261), and 1ul total-seq C hashtag antibody (C0301-C0310, Biolegend) and incubated for 30 minutes at 4°C.  Following wash, all samples were combined, resupended in sort buffer, and run on the cell sorter. To isolate Tregs, we sorted Dump-TCRb+CD4+Foxp3+ cells into catch buffer (1XPBS/50% FBS).  Cells were then centrifuged and resuspended in 1X PBS/10% FBS for single-cell capture. For 10X Genomics scRNA-seq, we generated three libraries that measure: (1) mRNA transcript expression (GEX), (2) sample/tissue-specific hashtag oligos (HTO) and protein marker levels using antibody derived tags (ADT), and (3) T cell receptor specific mRNA reperitorires. Cells were harvested and stained as described above. The sample was split into three libraries (GEX, HTO/ADT, and TCR). Reverse transcription PCR and library preparation were carried out under the Chromium Single Cell 5' v2 protocol (10X Genomics) per manufacturer's recommendations. The libraries were sequenced on the NovaSeq 6000 with 2x150 bp paired-end reads (Illumina). single cell RNAseq and immune reperitoire 10X Genomics