Supervilo cdna synthesis kit

1 × 10⁶ of T_H0, T_H1 or T_H2 cells were pelleted down by centrifugation at 200g, at 4°C for 5 min. Supernatants were discarded and cell pellets were immediately resuspended in 750 μl of Trizol (REF: 15596026; Ambion). Samples were snap-frozen in liquid N₂. For RNA isolation, cell lysates were thawed, and chloroform added (200 μl per 1 ml of Trizol). RNA samples were extracted as per the manufacturer’s instruction. The samples were vortexed for 20 s and incubated at room temperature for 3 min. The samples were pelleted down by centrifugation at 12,000g at 4°C for15 min. The top aqueous phase containing RNA was transferred into a new tube. RNA samples were purified by using Zymogen RNA clean and concentrator kit (R1017; Zymo research), as per the manufacturer’s instructions. RNA concentrations were obtained on a NanoDrop Spectrophotometer and 1.2 μg of RNA were subjected to cDNA synthesis by using the Super VILO cDNA synthesis kit (REF:11756050; Thermo Fisher Scientific). qRT–PCR was performed using Power up SYBR green master mix (REF: A25742; applied biosystems) on the QuantStudio three Real-Time PCR Systems, accordingly to the manufacturer’s instructions. The following gene expression levels analysed by qRT–PCR are listed in Table 1.