title: Cortisol (Hydrocortisone) Treatment of Activated Primary Human CD8⁺ T Cells
design: Cortisol (Hydrocortisone) Treatment of Activated Primary Human CD8⁺ T Cells
library_construction_protocol: Peripheral blood was obtained from healthy adult donors following informed consent and institutional ethical approval. Whole blood was processed immediately for PBMC isolation by density-gradient centrifugation using Ficoll-Paque Plus. CD8⁺ T cells were purified from PBMCs by immunomagnetic negative selection (STEMCELL Technologies). Purified CD8⁺ T cells were cultured in ImmunoCult-XF T Cell Expansion Medium supplemented with gentamicin (50 µg/mL) for a total of 8 days. Cells were activated for 72 h with plate-bound anti-CD3 (BioLegend, OKT3, 1 µg/mL) and anti-CD28 (BioLegend, CD28.2, 1 µg/mL), rested/expanded for 72 h in fresh medium containing human IL-2 (PeproTech, 100 U/mL), then re-stimulated for 48 h with plate-bound anti-CD3/anti-CD28 in the presence of hydrocortisone (Sigma; 100 nM) or vehicle control (methanol; Sigma). At the end of treatment, cell pellets were overlaid with 40 µL RNAlater (Invitrogen) and stored at −80°C prior to RNA extraction. Total RNA was extracted from frozen RNAlater-treated CD8⁺ T-cell pellets. Samples were homogenised using QIAshredder (Qiagen) according to the manufacturer's instructions. RNA was purified using the RNeasy Plus Mini Kit (Qiagen), including genomic DNA removal via gDNA Eliminator columns. RNA was eluted in RNase-free water and used for downstream library preparation. RNA-seq libraries were generated by Novogene using their standard mRNA-seq workflow. Briefly, poly-A–enriched mRNA was isolated, fragmented, and reverse-transcribed to cDNA, followed by end repair, A-tailing, adaptor ligation, and PCR amplification. Libraries were purified, quantified, and assessed for size distribution prior to sequencing.