# _Saccharomyces cerevisiae_'s Response to Dysprosium Exposure
## 2 Materials and Methods
### Gene Expression Analysis Through mRNA-Seq  
Total RNA was extracted from 5 \(\times\) 10\({}^{7}\) yeast cells cultured for 18 and 24 h in GYP with or without Dy(CH\({}_{3}\)COO)\({}_{3}\) using a Yeast Processing Reagent for total RNA preparation (Takara Bio, Kusatsu, Japan). The RNA was purified using NucleSpin RNA (Takara Bio). Reverse transcription and genomic DNA elimination were performed using the PrimeScript TM RT Reagent Kit with gDNA Eraser (Perfect Real Time) (Takara-Bio). The quality of RNA samples was evaluated by electrophoresis them using an Agilent 4200 TapeStation (Agilent Technology, Santa Clara, CA, USA) according to the High-Sensitivity RNA protocol provided by the manufacturer. The degree of degradation of total RNA was evaluated by checking the RNA integrity number equivalent (RINE), which is a value calculated based on the state of rRNA by the Agilent TapeStation using a maximum score of 10 to indicate the degree of RNA degradation (recommended value is 8 points or more), and samples with a score of 8 points or more were used. For the next-generation sequencing, the library was prepared from 4 ug of total RNA using the TrueSeq stranded mRNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Comprehensive sequencing was performed by MiSeq (Illumina); approximately 240 million reads were obtained from each sample (the procedure is detailed in [3]). The provided sequence reads were mapped to _S. cerevisiae_ S288C reference genome sequences (NCBI dataset, National Library of Medicine, Bethesda, MD, USA) using HISAT2 software (v2.0.1) (Kim Lab, Bioinformatics Department, University of Texas Southwestern Medical Center, Dallas, TX, USA), and quality control checks on raw sequence reads were performed using FastQC (Babraham Institute, Cambridge, UK). Functional annotation of the sequence reads obtained from the Saccharomyces Genome Database ([[https://www.yeastgenome.org/](https://www.yeastgenome.org/)]([https://www.yeastgenome.org/](https://www.yeastgenome.org/))) was performed using BLAST (accessed on 10 October 2020) searches of _S. cerevisiae_ annotation information. Additionally, differentially expressed genes (DEGs) of samples at each culture of different durations were identified using StringTie (Center for Computational Biology, Johns Hopkins University, Baltimore, MD, USA), and they were converted using the prepDE.py program (Center for Computational Biology), with a \(p\) value of <0.05 being identified as significant. The gene expression differences were presented in TPM (transcripts per million). The length of amplicons for tested and reference genes was 100-300 bp. Lastly, the functions of the extracted DEGs were determined and classified based on gene ontology (GO) biological process categories reported in the Saccharomyces Genome Database. Analysis of Gene Expression Ratio (2 - \(\Delta(\Delta\)Ct)) in Dy-Absorbing Culture Through Quantitative Real-Time Polymerase Chain Reaction (qRT PCR)  
Total RNA, extracted and qualified as described in the previous subsection, was reverse transcribed, while genomic DNA was excluded using the procedure also outlined in the previous subsection. The prepared cDNA was diluted 10-fold and utilized as a template for quantitative real-time PCR (SYBR Green Master Mix, Toyobo, Osaka, Japan) with the StepOne(tm) real-time PCR system (Thermo Fisher Sci., Tokyo, Japan) with targeted gene-specific primers (Table S1). A two-step PCR program was performed using the following protocol: DNA dissociation was performed at 95 \({}^{\circ}\)C for 1 min, primer annealing and extension were performed at 60 \({}^{\circ}\)C for 30 s, and all steps were repeated for 30 cycles. The delta cycle threshold (\(\Delta\)Ct) method [10] was employed to compare the relative expression of the target genes (Ct-TG) via the relative expression of the housekeeping gene mRNA (Ct-HK).