GSM9464024: rLCMV-D14-TCR; Mus musculus; OTHER
Murine spleens were isolated and mechanically disrupted using a metal mesh and a syringe plunger, and cells were counted with a Immunospot S6 device (C.T.L.). CD8 T cells were enriched by magnetic-activated cell sorting using the CD8+ T cells Isolation Kit, mouse (STEMCELL), but the antibody mix in the kit was replaced by a mix containing the following biotin-conjugated antibodies from BioLegend: anti-B220 (RA3-6B2), anti-CD19 (6D5), Ly-76 (TER-119), anti-CD4 (H129.19) and anti-CD138 (281-2). Cell suspensions were stained with antibodies against CD45R/B220 (RA3-6B2), CD8 (53-6.7) and DAPI purchased from BioLegend, PE-conjugated H2-Kb tetramers loaded with the SARS-CoV-2 spike epitope VNFNFNGL (produced by the University of Lausanne Tetramer core facility).  Cells were stained with oligo-conjugated antibodies (ADT) for surface markers using TotalSeq-C0250 anti-KLRG1 (138433, BioLegend), TotalSeq-C0563 anti-CX3CR1 (149043, BioLegend), TotalSeq-C0198 anti-CD127 (104455, BioLegend) and TotalSeq-C0112 anti-CD62L (135047, BioLegend) . Each condition included 3 biological replicates which were labelled with hashtag oligos (HTOs): TotalSeq-C0301 (155861, BioLegend), TotalSeq-C0302 (155863, BioLegend) or TotalSeq-C0303 (155865, BioLegend) before being pooled Tetramer-binding CD8 T cells were FACS-sorted (FACS Aria II, BD) and the recovered cells were immediately processed for cell capture and library preparation Chromium Next GEM Single Cell 5' Reagent Kits v2 (Dual Index) with Feature Barcode technology for Cell Surface Protein & Immune Receptor Mapping (CG000330 Rev D)