GSM8634668: CD4-TCell_LCMV_GEX; Mus musculus; RNA-Seq
Single cell suspension of splenocytes was obtained by mechanical disruption of spleen from infected mice with a syringe plunger over a 70um filter. CD4 T-cells were enriched by negative selection using biotinylated antibodies against CD8, B220, and TER-119 and Mojosort Streptavidin Nanobeads according to the manufacturers instructions (Biolegend). CD4 enriched cells were stained for 30 minutes at 4C with fixable viability dye (Tonbo)  n dPBS, 1 hour at 37C with I-Ab GP66-77 tetramer in 10% RPMI, and 30 mins at 4C with antibodies against CD4, PD1, and TotalSeqB (Biolegend) in FACS buffer (dPBS  + 1% BSA/  + 0.12% Sodium Azide). Virus-specific CD4 T-cells were FACS sorted on a Sony SH800z by expression of CD4 and GP66 tetramer. Sorted cells from each sample were combined in roughly equal numbers Chromium GEM-X Single Cell 3' Kit v4 according to the manufacturers instructions (10x)