RNA Extraction and Capture from Stimulated T Cells

RNA was prepared from stimulated PBMC stored at −70 °C in RLT buffer (Qiagen), using the RNeasy Mini Kit (Qiagen, Hilden, Germany) and following the manufacturer’s instructions. Cells were homogenized with QIAshredder columns (Qiagen). After extraction, RNA was quantified using a NanoDrop spectrophotometer (Thermo Fisher Scientific). RNA samples were stored at −70 °C. RNA was captured from stimulated CD4+ or CD8+ T cells stored at −20 °C in RNAlater (Thermo Fisher Scientific) after FACS sorting, according to a published protocol^{35 (link)}. Briefly, samples were mixed with lysis buffer (0.3% (v/v) Triton X100, 20 mM DTT, 2 mM dNTPs) in ratio 1:1 and vortexed. Magnetic Dynabeads (M-270 Streptavidin, Thermo Fisher Scientific) coated with RNA capturing polyT-tailed Indexing Oligonucleotides (Integrated DNA Technologies) were added to each well. After 5 min of incubation the magnetic beads were separated from the supernatant and washed twice with 6X SSC buffer. Subsequently, the beads were combined with RT mix as described later.