GSM9155534: 30Cto37C_100mMNAIN3_bioreplicate2_techreplicate1; Candida albicans; OTHER
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. We used 600ng of in vitro transcribed (IVT) RNA for library preparation following the manufacturer's protocol for the direct RNA library preparation kit SQK-RNA004 from Oxford Nanopore Technologies. The libraries were loaded onto FLO-PRO004RA PromethION RNA flow cells and sequenced on a PromethION 2 Solo device.The library preparation steps were the same as those for SQK-RNA002 except the following: 1) 1μl of SUPERase·In™ RNase Inhibitor (Invitrogen, AM2694) is added during the RT Adapter ligation reaction, 2) 6μl of RNA Ligation (RLA) instead of 6μl of RNA Adapter (RMX) is added in the second adapter ligation reaction, 3) the adapter-ligated RNA is eluted in 33μl of RNA Elution Buffer (REB), and 4) we prepared the sample for loading by adding 100μl of Sequencing Buffer (SB) and 68μl of Library Solution (LIS) to 32μl of the library sample.