GSM8362548: Donor1_DMSO_HT SS3_Plate11_832; 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 live and single CD4+ T-cells were sorted into 96-well plates containing lysis buffer using SH800S cell sorter (Sony Biotechnology Inc, San Jose, CA) with a 100-μm nozzle. The Smart-seq3 library preparations were performed as previously described [Hagemann-Jensen et al., Nat Biotechnol, 2020, 38:708-714] with some modifications. In addition, we developed and optimized an automated sample handling process for most steps of the Smart-seq3 protocol, including single-cell lysis, reverse transcription, and library construction by using Integra Biosciences VIAFLO 96 electronic pipetting system and Mantis liquid handler (Formulatrix, Bedford, MA, USA).