Prepare reagents according to recipes 1. Note that everything is in DEPC water. Inoculate W303a cells expressing different TOR1-RR variants in 2 mL SC medium overnight. 2. Subculture the cells starting from OD600=0.1 in 10 mL SC media, shake vigorously at 30 °C, 300 RPM for around 4-6 h until OD600=0.4-0.5. 3. Collect the cells by spinning down without freezing on ice. Discard supernatant. 4. Re-suspend cells with 1 mL water and transfer to a 1.5 eppendorf tube, quickly spin down at 3,000 x g for 15 sec. 5. Re-suspend cell pellet in 400 µL of AE buffer at room temperature. 6. Add 40 µL 10% SDS (final around 1%) and vortex briefly at room temperature (RT). 7. Immediately add 500 µL hot phenol/AE (put in 65 °C for 10 min before use), vortex vigorously for 1 min. Incubate at 65 °C for 5 min. Briefly vortex every 30 sec. 8. Immediately freeze by dumping into liquid nitrogen (labels won’t get off). Wait to thaw at RT (put in 30 °C to thaw may crack the tube). 9. Centrifuge for 10 min on a standard laboratory microfuge at 20,000 x g at RT. 10. Transfer around 400 μL supernatant to a new eppendorf tube. Recycle the lower phenol fraction carefully following the chemical safety protocol in your laboratory. 11. Add equal volume (400 μL) phenol: CHCl3/AE-Na. Vortex vigorously for 1 min at RT. 12. Spin down at 20,000 x g for 5 min in a standard laboratory microfuge. 13. Transfer supernatant (around 350 μL) to a fresh 1.5 mL eppendorf tube. Add CHCl3: isoamyl alcohol (24:1). Vortex vigorously for 1 min at RT. 14. Transfer aqueous supernatant to a fresh 1.5 mL microfuge tube. If white cloudy precipitate is observed between the aqueous phase and organic phase, repeat steps 17-18. 15. Add 1/10 volume of 3 M NaOAc (pH 5) and vortex vigorously. Add 2.5 volumes of ethanol. Vortex again. 16. Place at -20 °C for at least 30 min. 17. Spin down in the microfuge at 20,000 x g, 15 min at 4 °C. RNA pellet is usually visible. 18. Add ice-cold 75% EtOH, place at 4 °C for around 10 min. Vortex and spin down on microfuge 20,000 x g, 15 min at 4 °C. Discard supernatant. Suck out the liquid droplets in the tube. The white RNA pellet will turn clear when it dries out. Add 30-50 µL ddH2O (DEPC) immediately after it becomes clear. Do not let the RNA over-dry, which will make it difficult to dissolve. If RNA pellet is over-dry, dissolve RNA at 37 °C for 30 min. Store RNAs at -80 °C for more than 2 months.