RNA-seq of Saccharomyces cerevisiae, Lachancea thermotolerans, Torulaspora delbrueckii: direct cell contact
The pre-processing of mRNA for sequencing and subsequent data analysis performed in our study were similar to those previously reported by Conacher et al. (2022), who also made use of the services provided by the Central Analytical Facility for Next Generation Sequencing at Stellenbosch University. In summary, a Bioanalyzer 2100 (Agilent Technologies, Waldbronn, Germany) with RNA 6000 Nano Chip and reagents were used to determine RNA integrity number [RIN] values and quantity of total RNA in extraction samples, after which 800 ng total RNA was used to isolate polyadenylated (poly(A)) mRNA using a Dynabeads mRNA Direct Micro kit (ThermoFisher Scientific) according to the manufacturers instructions. Hereafter, a representative cDNA library was prepared by the conversion of expressed mRNA transcripts using an Ion total transcriptome sequencing (RNASeq) kit v2 (ThermoFisher Scientific), after which library purification was performed while also assessing yield and fragment properties using an Agilent Bioanalyzer 2100 using the high-sensitivity DNA chip and kit (Agilent Technologies). Thereafter, library concentrations were adjusted to 80 pM before being pooled in equimolar amounts for template preparation using an Ion 540 Chef kit (ThermoFisher Scientific). This was followed by strand-specific massively parallel sequencing on an Ion Torrent GeneStudio S5 Prime platform using a standardized sequencing protocol and reagents. Default parameters in the Torrent Suite version 5.12.2 software were used for flow space calibration and basecaller analysis.