Tumor Tissue Preparation and RNA Isolation

Tumors were sliced into thin sections manually with a sharp surgical blade. Sections were then incubated in RPMI media containing 10% FBS, L-glutamine, sodium pyruvate, penicillin/streptomycin, HEPES, nonessential amino acids, and beta-mercaptoethanol on 24-well polycarbonate Transwell inserts with a 0.4 μm pore size (Corning) and maintained in 5% CO₂ and atmospheric oxygen levels [18 (link)]. Tissues were incubated with viral peptides at 10 μg/mL or in equal volume of DMSO for 9 h. Tissues with poor viability after culture were excluded. Tumor sections were stored in RNAlater (ThermoFisher) at 4 degrees overnight, then stored at −80 until further processing. For RNA isolation, tissue was thawed on ice in 1 mL TRIZOL (Invitrogen), then homogenized with a Tissue Tearor homogenizer, BioSpec. RNA was then isolated following the TRIZOL recommended protocol. Resulting RNA was then further purified using Qiagen RNA Cleanup Kit. Peptides used in human studies: CLGGLLTMV (EBV_CLG), GLCTLVAML (EBV_GLC), NLVPMVATV (CMV_NLV), GILGFVFTL (IAV_GIL).