GSM6138874: 1_ΔΔ_SD-HS_t90; Candida albicans; RNA-Seq
GSM6138874
For total RNA isolation cells were resuspended in 440 µl AE buffer with 10% SDS and 440 µl of Acid-phenol:Chloroform with isoamyl alcohol (Ambion, Germany) was added. The suspension was thoroughly mixed and 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 centrifugation at 16000 g, the upper aqueous phase transferred and 1/10 of the volume sodium acetate (pH 5.3) and 1 volume of 2 propanol was added to precipitate the RNA for at least 30 min at -20°C. The supernatant was discarded after 10 min centrifugation at 12000 g, the RNA pellet washed twice with 80 % ethanol and finally solved in 300 µl nuclease free H2O. Quality and quantity of the RNA was assessed using 2100 Bioanalyzer (Agilent Technologies, California, USA) and Nanodrop (Thermo Fischer Scientific, Massachusetts, USA) measurements. 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® UltraTM RNA. Library Prep Kit for Illumina® (NEB, USA) following manufacturer's recommendations and index codes were added to attribute sequences to each sample. Briefly, mRNA was purified from total RNA by mRNAusing poly-T oligo-attached magnetic beads. Fragmentation was carried out by Covaris sonication. First strand cDNA was synthesized using random hexamer primer and M-MuLV Reverse Transcriptase (RNase H-). Second strand cDNA synthesis was subsequently performed using dTTP. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities. After adenylation of3' ends of DNA fragments, NEBNext Adaptor with hairpin loop structure were ligated to prepare for hybridization. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system (Beckman Coulter, Beverly, 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 are assessed by Qubit and real-time PCR. Sequencing platform was Illumina Novaseq 6000 PE150. Paired end RNA sequencing of cDNA fragments of preferentially 150~200 bp in length.