title: Illumina NovaSeq 6000 sequencing; Type I interferon signaling in fibroblastic reticular cells prevents exhaustive activation of antiviral CD8+ T cells
design: Type I interferon signaling in fibroblastic reticular cells prevents exhaustive activation of antiviral CD8+ T cells
library_construction_protocol: Inguinal lymph nodes were harvested, collected into RPMI 1640 medium containing 2% FCS, 20mM Hepes (all from Lonza), 0.375 mg/ml Collagenase P (Sigma Aldrich) and 100 μg/ml DNase1 (Applichem) and shred into small pieces using 22 guage needles. Samples were incubated at 37C for 60 min, with resuspension and collection of the supernatant every 15 min into PBS containing 0.5% FCS and 10mM EDTA. Stromal cells were enriched by depleting CD45+ hematopoietic cells and Ter119+ erythrocytes using MACS microbeads (Miltenyi). Mice were subcutaneously infected in one flank with 200 ffu of lymphocytic choriomeningitis virus  (LCMV) Armstrong. Analyses of EYFP+ reticular cells were performed at day 3 after infection. LCMV titrations/CD8+ T cell responses were performed at different time points after infection. EYFP+ reticular cells from lymph nodes of naïve or infected Ccl19-EYFP or Ccl19-EYFP Ifnarfl/fl mice were sorted using the BioRad S3 Cell Sorter. Single cell suspensions were run on the 10x Chromium (10x Genomics) system. The cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v2 Chemistry)