Bulk RNA-seq OT-I Cd8 T cells
Bulk RNA-seq OT-I Cd8 T cells
CD8+ T cells were purified from spleens of OT-I mice by negative selection with magnetic beads (EasySep, Stemcell Technologies). After purification, cells were >95% CD8+ T cells. In each well of a 96-well plate, 0.5x106 of the purified CD8+ T cells/ml were cultured in complete media (RPMI 1640, 10% FBS (Gibco), 1% 2 mM L-glutamine (Life Technologies), 1% HEPES (Life Technologies), 1 nM Sodium Pyruvate (Life Technologies), 1% non-essential amino acids (Life Technologies), 100 U/ml penicillin (Gibco) and 100 μg/ml streptomycin-sulfate (Gibco), 0.05 mM 2-mercaptoethanol (Sigma) with recombinant human IL-2 (10 ng/ml, Roche) with or without 10 ng/ml OVA(257–264) (SIINFEKL) peptide in the presence or absence of 25 ng/ml murine IL-27 (R&D Systems). For repeated peptide stimulation, 10 ng/ml OVA(257–264) peptide was added daily for 4–6 days. Cells were checked daily, passaged, and cultured with fresh complete media containing cytokines as required. After day 5 cells were washed and stained for flow cytometry. RNA was extracted with RNeasy Mini Kit (Qiagen) for RNA sequencing. Total RNA was quantified with Qubit RNA HS Assay Kit (Thermo Fisher Scientific) and quality was assessed using RNA ScreenTape on 4200 TapeStation (Agilent Technologies). For OT-I T cell sequencing library generation, the Truseq Stranded mRNA kit (Illumina) was used with an input of 100-1000 nanograms of total RNA. Libraries were quantified with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) and the average library size was determined using D1000 ScreenTape on 4200 TapeStation (Agilent Technologies).