title: GSM8992175: Untreated Wt Rep3; Mus musculus; RNA-Seq
library_construction_protocol: A total of 2–5 × 10⁵ cells per condition were lysed in 0.5 mL of TRIzol Reagent (Thermo Fisher) following the indicated treatments. Total RNA was extracted, and RNA-seq analysis was performed by the Genome Information Research Center (GIRC) at Osaka University. cDNA libraries were prepared using the TruSeq Stranded mRNA Sample Prep Kit (Illumina, Cat. No. 20020594) according to the manufacturer's instructions. Sequencing was conducted on an Illumina HiSeq 2500 platform using 75-base single-end reads. Base calling was performed with Illumina Casava v1.8.2 software. Sequenced reads were aligned to the mouse reference genome (mm10/GRCm38) using TopHat v2.0.13 with Bowtie2 v2.2.3 and SAMtools v0.1.19. Gene expression levels were quantified as fragments per kilobase of exon per million mapped reads (FPKM) using Cuffnorm v2.2.1. Further data processing and analysis were conducted on the Galaxy platform (https://usegalaxy.org/). Read quality was assessed using FastQC (v0.73), and adaptor sequences were trimmed prior to alignment with HISAT2 (v2.2.1) using default parameters. Gene counts were generated with featureCounts (v2.0.3), and differential expression analysis was performed using DESeq2 (v2.11.40.7). Visualization of results, including volcano plots and heatmaps, was performed using Volcano Plot (v0.0.5) and heatmap2 (v3.1.3), respectively. Gene ontology enrichment and pathway analysis were conducted using the PANTHER GO-Slim Biological Process database (v17.0, released 2022/02/22).