title: RNA-seq of Candida albicans cells of the wildtype strain SC5314 and a sky2 deletion mutant grown on different carbon sources (glucose, malic acid, succinic acid)
design: RNA-seq of Candida albicans cells of the wildtype strain SC5314 and a sky2 deletion mutant grown on different carbon sources (glucose, malic acid, succinic acid)
library_construction_protocol: C. albicans was pre-grown overnight at 30°C in  SD (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 2% glucose) and used to set cultures in Glucose (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 1% glucose), Malic acid ((0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 1% malic acid) and succinic acid (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 1% succinic acid) medium at OD600 0.2. Cells were incubated for 4 h (glucose) or 6h (malic acid, succinic acid) at 37°C and shaking at 180 rpm. Cells were separated from test medium by centrifugation for 4 min at 4000g and 4ºC. Cells were shock frozen in liquid nitrogen and stored at -80°C until further processing. C. albicans was pre-grown overnight at 30°C in  SD (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 2% glucose) and used to set cultures in Glucose (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 1% glucose), Malic acid ((0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 1% malic acid) and succinic acid (0.17 yeast nitrogen base w/o ammonium sulfate and w/o amino cids, 0.5% ammonium sulfate, 1% succinic acid) medium at OD600 0.2. Cells were incubated for 4 h (glucose) or 6h (malic acid, succinic acid) at 37°C and shaking at 180 rpm.  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 -80°C for 30 min and subsequently thawed at 65°C for 5 min. The phases were separated via 4 min centrifugation at 13,000 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 overnight at -20°C. The supernatant was discarded after 10 min centrifugation at 12,000 g, the RNA pellet washed twice with 70 % 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 0.5 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. 