Determining the impact of escins on Saccharomyces cerevisiae ergosterol biosynthesis mutants

This RNAseq data was collected in order to determine the impact of treatment with the triterpenoid saponin mixture escin (also known as aescin) on the transcriptomes of wild type yeast, and mutants in ergosterol biosynthesis. The study used Saccharomyces cerevisiae BY4741 as the wild type background strain, and deletion mutants (erg6? and erg3?) from the Yeast Deletion Collection. Overnight cultures in YPD media were used to inoculate 20 mL YPD in 100 mL conical flasks at OD600 0.1. Cultures were grown (30 degrees C, 200 rpm shaking) to mid-log phase, and then treated with 0, 75 or 100 ug/mL escin in water for 60 mins. Cell pellet samples of approximately 2 x 107 cells were frozen in liquid nitrogen and stored at -80 °C prior to RNA extraction. At the University of Liverpool Centre for Genomic Research, total RNA samples were treated concomitantly with DNase (Turbo DNA-freeTM kit; AM1907; Ambion by Life Technologies) and SUPERase•In RNase Inhibitor (AM2694; Invitrogen). After treatment, the samples were purified using Agencourt RNAClean XP beads (A66882; Beckman-Coulter). rRNA depletion was carried out by use of the NEBNext® rRNA Depletion Kit (E7850L; New England BioLabs), but substituting the bacterial baits with yeast baits. NEBNext Ultra II Directional RNA libraries were prepared from rRNA depleted RNA (E7760L; New England BioLabs), and paired-end RNA-Seq reads were generated using an Illumina NovaSeq 6000. For data analysis, first raw FASTQ files were trimmed for the presence of Illumina adapter sequences (wherever the 3' end matched the adapter sequence for =3 bp) using Cutadapt (v1.2.1). Reads were further trimmed using Sickle (v1.200), with a minimum window quality score of 20, and reads <10 bp after trimming were removed. Additional read quality checks were performed using FastQC (v0.11.9) and MultiQC (v1.10). Reads were then mapped to a representative genome annotation for BY4741 (http://sgd-archive.yeastgenome.org/sequence/strains/BY4741/BY4741_Toronto_2012/) from the Saccharomyces Genome Database, using the universal RNA-sequence aligner, STAR (v2.7.7a). Resultant BAM files were quality-filtered, using a mapping quality (MAPQ) cut-off of 244. Quality filtered files were then assessed for quality using the depth utility of Samtools (v1.9). Quality-assessed BAM files were then used with the genome annotation for BY4741, to generate a table of aligned read counts, using featureCounts (v2.0.1) from the subread package, with the minimum number of overlapping bases in a read required for read assignment set to 150 bp. Key words: Escin, aescin, triterpenoid, saponin, ergosterol, sterol, yeast, Saccharomyces cerevisiae Collection date: 8th January 2020 Strain: Saccharomyces cerevisiae BY4741