GSM9130721: 37C_40min_Rep2; Candida albicans SC5314; RNA-Seq
To extract RNA from C. albicans, we first thawed the frozen pellet at room temperature and then placed it on ice. The pellets were re-suspended in 2.8ml yeast lysis buffer (100ml lysis buffer: 0.7ml 1M Tris-HCl, pH 7.0, 0.3ml 1M Tris-HCl, pH 8.0, 2ml of 0.5M EDTA, 5ml of 10%SDS). We transferred 700μl of this mixture into a 1.5ml Eppendorf tube, added 700μl of acid phenol, and vortexed rigorously. We then incubated the tubes at 65°C in a thermomixer for one hour, with constant shaking at 1000 rpm. After the incubation, we centrifuged the sample at maximum speed for 10 minutes at 4°C. We then transferred the aqueous phase into a new tube, added the same volume of fresh acid phenol, vortexed it, and spun it at maximum speed for 10 minutes at 4°C. We performed acid phenol extraction at least 2-3 times until the aqueous phase looked clear. To precipitate the RNA from the aqueous phase, we transferred the aqueous phase into a 2 ml tube and added 1/10 volume of 3M sodium acetate and 3x ice-cold 100% ethanol. We precipitated the RNA at -20°C for 1 hour before spinning it at maximum speed for 20-30 mins at 4°C. The pelleted RNA was then washed in 800μl of ice-cold 70% ethanol, centrifuged to remove the ethanol, and then air dried for 2 min before being dissolved in 50μl of nuclease-free water. The RNA half-life was measured as previously described. Briefly, C. albicans (sc5314) was inoculated into 10ml of liquid YPD media and grown overnight shaking at 30°C and 250rpm. Aliquot the overnight cultured Candida albicans cells to fresh medium (1:100), and continue culture at 30°C, 250rpm for around 3 hours until OD600=0.6. The aliquot Candida albicans culture was then centrifuged and resuspended in 30°C or 37°C prewarmed YPD medium. The Thiolutin (Merck, CAS RN 87-11-6) was then added to each aliquot to a final concentration of 10μg/ml. The culture aliquots were then cultured for 20min, 40min, 60min, and 120min at 30°C or 37°C, respectively. The cells were collected, and the RNA was extracted as described above, and the RNA library was then prepared using Illumina Nextra library prep kit. The library is sequenced on a NEXTSEQ-HIGH to generate 1X76 base reads.