Illumina NovaSeq 6000 paired end sequencing; RNA-seq of high-lipid producing, no-lipid producing, and control Yarrowia lipolytica strains on media with high and low carbon/nitrogen ratios, with ammonium sulphate or urea as nitrogen sources, in chemostats with two different dilution rates.
RNA-seq of high-lipid producing, no-lipid producing, and control Yarrowia lipolytica strains on media with high and low carbon/nitrogen ratios, with ammonium sulphate or urea as nitrogen sources, in chemostats with two different dilution rates.
Samples for RNA extraction were taken after at least four retention times of steady-state fermentation by rapidly withdrawing 10 ml of culture and injecting it into a 50 ml falcon tube with ca. 35 mL of crushed ice. The samples were immediately centrifuged at 4000 rpm for 5 min at 4°C. The supernatant was discarded, the pellet was resuspended in ice-cold water, transferred into 2 mL reaction tubes, and centrifuged at 4000 rpm for 5 min at 4°C. The supernatant was removed, and the open tubes were tapped on paper towels to remove the remaining residue. Dry samples were immediately frozen in liquid nitrogen and stored at −80 °C until further analysis.  Chemostat fermentations were performed in DasGip 1-L stirrer-pro vessels (Eppendorf, Jülich, Germany), with a working volume of 500 mL at 28 °C, 600 rpm agitation and aeration at 1 vvm (= 30Lh-1). pH was monitored with a pH sensor (Mettler Toledo, Switzerland), and pH was maintained at 5.0 by automatic addition of 2M KOH. Cells were grown in batch, and their growth was monitored via the CO2 exhaust gas. After leaving the exponential growth phase, the constant feed was initiated to obtain a steady-state cultivation with a dilution rate of 0.1 h−1 or 0.06 h-1. The working volume of 500 mL was kept constant using an overflow pump. The samples for transcriptome analysis were taken after at least 4 residence times of steady-state growth. Each strain was fermented in at least 3 reactors. The RNA extraction was performed using the RNeasy Mini Kit (QIAGEN). The DNA was removed from the sample using RNase-Free DNase Set (QIAGEN). The quality of purified RNA samples was analyzed with a 2100 Bioanalyzer (Agilent Technologies, Inc., Santa Clara, CA) and stored at −80°C until further analysis. The RNA library was constructed using Illuminar TruSeq Stranded mRNA.