Hisequation 2500 system

Total RNA (1 μg) was thawed to create a library using TruSeq Stranded RNA LT Guide (Illumina, San Diego, CA, USA) according to the manufacturer's instructions. An Agilent 2100 bioanalyser (Santa Clara, CA, USA) was used to evaluate the concentration and size distribution of complementary DNA (cDNA) in the library before sequencing with the Illumina HiSequation 2500 system. The high-throughput sequencing was performed according to the manufacturer's instructions (Illumina HiSequation 2500 User Guide).
The raw data were filtered by FASTX (ver. 0.0.13) before mapping to the genome using TopHat (ver. 2.0.9). Gene fragments were counted using HTSeq followed by trimmed mean of M values (TMM) normalization. Significantly differentially expressed genes (DEGs) were identified using Cufflinks (ver. 2.2.1) [12 (link)]. DEGs were then submitted to Visualisation and Integrated Discovery analysis (DAVID; ver. 6.8) [13 (link)] for gene ontology (GO) term enrichment and clustering and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway mapping using default parameters, except for an EASE score setting of 0.05.

A starter of cells was grown overnight on SC medium containing 2% carbon source (glucose, galactose, or xylulose). The cells were then diluted to OD_600nm = 0.1 in 15 ml tubes and grown until they reached mid log phase (0.4 < OD_600nm < 0.6). Cells were then harvested by 1 min centrifugation at 4000 rpm, the supernatant was discarded, and they were frozen in liquid nitrogen.
RNA was extracted using the Nucleospin 96 RNA kit with modifications for working with yeast. Lysis was performed by mixing the cells with 450 µl lysis buffer [1 M sorbitol (Sigma S1876), 100 mM EDTA 0.5 M, and 100 U/ml lyticase]. The lysis mixture was transferred to a 96-well plate that was incubated at 30° for 30 min. The plate was then centrifuged for 10 min at 2500 rpm, and the supernatant was transferred to a 96-well plate provided by the Nucleospin 96 RNA kit, followed by extraction as described in the kit protocol.
Labeled cDNA was created from RNA extracts, and cDNA was barcoded and then sequenced in the Illumina HiSequation 2500 system, using a Truseq SR Cluster Kit v3 -cBot-HS cluster kit and a Truseq SBS Kit v3-HS run kit (50 cycles).

As described in Voichek et al.,(2018) (link). Briefly: Cells were grown to OD₆₀₀ of 0.2-0.4 after >6hr in exponential growth and flash-frozen in liquid nitrogen after centrifugation and media removal. RNA was extracted using the Nucleospin 96 RNA kit with modifications for working with yeast. Lysis was performed by mixing the cells with 300 µl lysis buffer [1M sorbitol (Sigma S1876), 100 mM EDTA 0.5 M, and 100 U/ml lyticase]. The lysis mixture was transferred to a 96-well plate that was incubated at 30° for 30 min. The plate was then centrifuged for 10 min at 3000 rpm, and the supernatant was transferred to a 96-well plate provided by the Nucleospin 96 RNA kit, followed by extraction as described in the kit protocol. Labeled cDNA was created from RNA extracts, and cDNA was barcoded and then sequenced in the Illumina HiSequation 2500 system, using a Truseq SR Cluster Kit v3 -cBot-HS cluster kit and a Truseq SBS Kit v3-HS run kit (50 cycles).