GSM7431409: H99_rapamycin 60min sample_2; Cryptococcus neoformans var. grubii H99; RNA-Seq
1. Freeze cells in Liquid Nitrogen for 30min. 2. Lyophilize samples overnight. 3. Add 3ml of 3mm glass beads and vortex vigorously until fine powder is created. 4. Add 4ml of Easyblue (dissolve powder completely) and let them sit 5 minutes. 5. Add 800 ul of chloroform (invert 10 times). 6. Let them sit 3 minutes and transfer to 14 ml round bottle tubes. 7. Spin at 10,000 rpm for 15 minutes. 8. Transfer supernatant to new round-bottom tubes. 9. Add 1.6 ml of isopropanol and capping and gently invert. 10. Store for 10 minutes at room temperature. 11. Spin for 10 minutes at 10,000 rpm. 12. Pour off supernatant. 13. Add 4 ml of 75% RNase free-EtOH (made with DEPC dH2O) and invert 4-5 times. 14. Spin for 5 minutes at 8,000 rpm. 15. Pour off EtOH (Let tubes air-dry upside down). 16. Resuspend in appropriate volume of DEPC dH2O. 17. Heat at 55℃ for 10 minutes. 18. Transfer RNA to microtube and measuring the concentration. 19. For purification of the extracted total RNA, we used RNeasy spin column (Qiagen) following the manufacturer's protocol. RNA libraries were prepared for sequencing using standard Illumina protocols.