GSM8529285: WT HQN III; Candida albicans; RNA-Seq
Total RNA was isolated from C. albicans cells using phenol-chlororform extraction as previously reported (https://doi.org/10.1128/mbio.03142-21): "For total RNA isolation, cells were resuspended in 440 μL acetate EDTA buffer (50 mM sodium acetate, 10 mM EDTA) with 10% SDS, and 440 μL of acid-phenol-chloroform with isoamyl alcohol (Ambion, Germany) was added. The suspension was thoroughly mixed, incubated for 5 min at 65°C, frozen at −20°C for 30 min, and subsequently thawed at 65°C for 5 min. The phases were separated via 4 min of centrifugation at 16,000 × g, the upper aqueous phase was transferred, and a 1/10 volume of sodium acetate (pH 5.3) and 1 volume of 2-propanol were added to precipitate the RNA for at least 30 min at −20°C. The supernatant was discarded after 10 min of centrifugation at 12,000 × g, and the RNA pellet was washed twice with 80% ethanol and finally solved in 300 μL nuclease-free H2O. The quality and quantity of the RNA were assessed using 2100 bioanalyzer (Agilent Technologies, CA, USA) and Nanodrop (Thermo Fisher Scientific, MA, USA) measurements." Sequencing libraries were prepared as previously reported (https://doi.org/10.1128/msystems.00539-22): "A total amount of 0.4 μg RNA per sample was used as input material for the RNA sample preparations. Sequencing libraries were generated using NEBNext Ultra RNA library prep kit for Illumina (New England Biolabs [NEB], USA) following the manufacturer's recommendations, and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA using poly(T) oligonucleotide-attached magnetic beads. Fragmentation was carried out by Covaris sonication. First-strand cDNA was synthesized using random hexamer primers and Moloney murine leukemia virus (M-MuLV) reverse transcriptase (RNase H−). Subsequently, second-strand cDNA synthesis was performed using dTTP. The remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. Afterward, the 3′ ends of DNA fragments were adenylated. Next, an NEBNext adaptor with hairpin loop structure was ligated for hybridization. To select cDNA fragments of preferentially 250 to 300 bp in length, the library fragments were purified with the AMPure XP system (Beckman Coulter, Beverly, MA, USA). Then, 3 μL User enzyme (NEB, USA) was used with size-selected, adaptor-ligated cDNA at 37°C for 15 min followed by 5 min at 95°C before PCR. Then, PCR was performed with Phusion high-fidelity DNA polymerase, universal PCR primers, and index (X) primer. At last, PCR products were purified (AMPure XP system), and library quality and quantity were assessed by Qubit and real-time PCR. The clustering of the index-coded samples was performed on a cBot Cluster Generation System using a cBot-HS PE cluster kit (Illumina) according to the manufacturer's instructions."