GSM8345785: Donor2_DMSO_Rep1_Bulk RNAseq; Homo sapiens; RNA-Seq
PBMCs from healthy donors were isolated by SepMateTM tubes (STEMCELL Technologies, Catalog#85450), following manufacturer's instruction, and resuspended in RPMI 1640 medium (Gibco, Catalog#11875093) containing 5% human serum (Sigma, Catalog# H3667-100ML) and 1% penicillin-streptomycin and cultured in 6-well plates (3 mL/well). After overnight culture, the PBMC was resuspended and cultured at 5 × 106 cells/ml in 48-well plate and treated immediately with DMSO or phorbol-12-myristate-13-acetate (PMA) plus ionomycin (PMA/Ionomycin) cocktail (Cell stimulation cocktail, eBioscience, Catalog#00-4970) for 3 h. Then, PBMC was stained with APC mouse anti-human CD4 (SK3, BD Biosciences, Catalog#340443) and Alexa Fluor 488 mouse anti-human CD3 (UCHT1, BioLegend, Catalog#300454) before sorting. The dead cells were gated out after staining with viability dye eFluor 780 (Invitrogen, Catalog#65-0865-14). The CD4+ T-cells were sorted into 15-ml centrifuge tubes with collection buffer (PBS with 1% BSA). Qiagen RNeasy Mini Kit was used for RNA extraction. RNA-seq libraries were generated using the TruSeq Stranded mRNA Kit