title: RNA-Seq of Festuca arundinacea, Low Frost Tolerant cultivar, day 21 of re-acclimation, biological sample 2
design: Total RNA was extracted from the samples (three biological replicates for time-point) using the RNeasy Mini Kit (QIAGEN, Germany), in accordance with the manufacturers protocol, and subsequently treated with RNase-Free DNase Set (QIAGEN) to remove any remaining genomic DNA. RNA quality and integrity were assessed in a two-step process. Initial evaluation was carried out using the Agilent 4200 TapeStation system with the RNA ScreenTape assay (concentration range: 25500 ng/L; fragment size: 506000 nucleotides). To confirm RNA integrity, a secondary quality assessment was performed using the Agilent 2100 Bioanalyzer. Minimal threshold of RNA integrity (RIN) value acceptance was 7. RNA sequencing was conducted by Macrogen Europe (headquartered in Seoul, South Korea) using the TruSeq Stranded mRNA Library Prep Kit (Illumina) and an Illumina sequencing platform. Sequencing was performed in a paired-end mode (2  151 bp). The analysis was based on the reference genome MPB_Lper_Kyuss_1697 and the annotation file GCF_019359855.1 After sequencing, trimmed reads were mapped to the reference genome (MPB_Lper_Kyuss_1697) using HISAT2, and the overall mapping ratio was evaluated. Transcript assembly was then performed for each sample using StringTie. Expression profiles for each gene and transcript were quantified as read counts, FPKM (Fragments per Kilobase per Million mapped reads), and TPM (Transcripts Per Kilobase Million). Differentially expressed genes (DEGs) were identified across 12 pairwise comparisons using DESeq2, applying thresholds of |fold change|  2 and raw p-value < 0.05