Bulk RNA sequencing of cytotoxic T lymphocytes of OT1 mice

We found that MC38 tumor-conditioned medium(TCM) treatment significantly impacted multiple pathways including cholesterol metabolism pathway of CTLs. Further analysis of cholesterol metabolism pathway, we observed that Cholesterol-25-hydroxylase(Ch25h) expression was significantly downregulated after treated with TCM compared to treated with fibroblasted-conditioned medium(FCM). In the meanwhile, we found that activating transcription fator 3(Atf3) was signifcantly upregulated after TCM treatment. Finally, we investigated the Atf3-Ch25h axis regulating CTLs function in tumor context. Splenocytes were isolated from WT OT1 mice and stimulated with OVA peptide for 48 hours followed by FCM or TCM treatment for 8 hours. After that, RNA was extracted using mini RNA isolation kit and concentration was measured using nanodrop2000.