Comprehensive RNA-seq Protocol for Differential Gene Expression Analysis

RNA quality was evaluated using Agilent Bioanalyzer 2100 Eukaryote Total RNA Pico chips. RNA-seq libraries were prepared using the SMART-Seq version 4 Ultra Low Input RNA Kit (Takara, 634889) from total RNA, following the manufacturer’s protocol. Libraries were then sequenced on a HiSeq 4000 using 75-bp paired end reads to an average depth of 22,445,650 ± 240,398 reads (SEM). Transcript abundance estimates were quantified using Salmon to mouse reference transcriptome from assembly GRCm38 (mm10), aggregated to gene level for UCSC-annotated genes using tximport, and DESeq2 was used to calculate normalized counts and differential expression [88 (link), 89 (link)]. CD4 and CD8 up/down gene signatures were generated through differential expression analysis via DESeq2. Differentially expressed genes (DEGs) between Yap-cKO versus WT CD4⁺ and CD8⁺ cells were defined as log2(FC) > 1 (up) or log2(FC) < −1 (down) and FDR < 0.05. DEGs were visualized with a heatmap combined with a barplot annotation, with the heatmap cells representing the log-normalized expression values for each sample. Each row is accompanied by a bar representing the log-fold change in gene expression (KO/WT). Plots were generated using the ComplexHeatmap software package available in R. Data have been deposited in NCBI GEO (accession number GSE139883).