title: GSM6745549: IL-12/18 Adult [scRNA-seq]; Mus musculus; RNA-Seq
library_construction_protocol: Murine cells were isolated from isolated from spleen and 4-6 neonatal tissues were pooled per biological sample. Single cell suspensions from spleen were obtained by manual dissociation and filtration through a 40uM strainer. CD8+ T cells were isolated by positive magnetic separation using anti-CD8a microbeads. 2x105 cells were plated per well in round-bottomed 96 well plates. Cells were incubated in RPMI supplemented with 10% fetal bovine serum, L-glutamine, penicillin-streptomycin, 2-mercaptoethanol (RP-10) and 10 ng/ml recombinant human IL-2 (Thermo Fisher Scientific) for 18-22 hours. For bystander-activated samples, media was additionally supplemented with recombinant murine IL-12p70 (Thermo Fisher Scientific) and recombinant murine IL-18 (Thermo Fisher Scientific) at 1 ng/ml. 1.5 mg/ml Brefeldin A (Millipore Sigma; St. Louis, MO) was added to the cells for the final 4 hours of incubation prior to antibody labeling for flow cytometry analysis. Live Va2+CD8+ cells were sorted into 0.04% BSA in PBS. Cells were counted and 3-8k cells per sample were loaded in the Chromium controller for the formation of gel bead-in-emulsions (GEMs) following the manufacturer's instructions (10X Genomics). The single-cell RNA-seq libraries were prepared using Chromium Single Cell 3′ Reagent Kit v2 following the accompanied protocol and were sequenced on Illumina NextSeq500.