GSM8742915: RV d7 CD4, GEX, biol rep 2; Mus musculus; RNA-Seq
Single cell suspension of pooled Peyer's patches (PPs) and mesenteric lymph nodes (MLNs) from rotavirus (RV) infected mice were stained with VP4:p:I-Ab or VP6:p:I-Ab tetramers and tetramer-binding cells were enriched by magnetic beads and the beads-unbound fractions were also collected. The bead-bound and unbound cells were stained for cell surface markers for flow cytometric sorting as well as hashtag oligonucleotides (HTOs) Abs. Each sample was stained with unique HTO-labeled antibodies to CD45 and MHCI (Biolegend, TotalSeq C anti-mouse Hashtag Abs): HTO1 (for MLN_VP4, M1/42; 30-F11, 155861), HTO2 (for MLN_VP6, M1/42; 30-F11, 155863), HTO3 (for PP_VP4, M1/42; 30-F11, 155865), HTO4 (for PP_VP6, M1/42; 30-F11, 155867) and HTO5 (for Total, M1/42; 30-F11, 155869). Then, by flow cytometric sorting, Dump (B220, CD11b, CD11c, F4/80 and viability dye)-negative CD3+CD4+CD8–p:I-Ab+ cells from the bead-bound fractions (referred as VP4:I-Ab+ and VP6:I-Ab+) and an equal number of Dump-negative CD3+CD4+CD8–p:I-Ab– cells from the bead-unbound fraction (referred as Total CD4+) were sorted into the same collection tube. The sorted cells were counted for viability and immediately subjected to library preparation using 10X Chromium Next GEM Single Cell 5' Reagent Kits V2 and Chromium Single Cell Mouse TCR Amplification Kit according to the manufacturer's instructions.