RNA sequencing of wild-type S. cerevisiae (BY4741) thermo-pulsed with optimal ( 30 °C- 30 °C) or elevated ( 30 °C- 37 °C) temperatures
RNA sequencing of wild-type S. cerevisiae (BY4741) thermo-pulsed with optimal ( 30 °C- 30 °C) or elevated ( 30 °C- 37 °C) temperatures
The yeast culture was given thermal stress and then centrifuged. Cells were divided into two groups for two different thermal pulsing conditions. In the first condition, cells were maintained at 30 °C in YPD broth for 15 minutes, followed by another 15 minutes at 37 °C, resulting in a single cycle of thermal pulsing (TP). In the second condition, a steady state (SS) was created when the cells were kept at 30 °C for both of the 15-minute time intervals. Using the aforementioned methods, twenty-five cycle pulsed cells and steady state cells were produced. RNA isolation was done using TRIzol Reagent. The sample was homogenized with TRIzol Reagent, followed by addition of chloroform. The solution was allowed to separate into different layers, and the upper aqueous layer was transferred to isopropanol, in the presence of glycogen for precipitation. The precipitated RNA was washed with ethanol, and resuspended in nuclease-free water. DNAse treatment was done to remove DNA contamination. The pure RNA was again precipitated with ethanol, washed and resuspended in nuclease free water for downstream processing. SPRI Beads-based Size Selection