title: RNA-Seq of myocardium: adult male mice
design: For RNA sequencing, samples from the HS-24h groups were analyzed (n = 3). Total RNA was extracted from mouse heart tissue using TRIzol reagent (Invitrogen, NY, USA). RNA concentration and quality were assessed by 1% agarose gel electrophoresis and an Agilent 2100 Bioanalyzer. Libraries with an insert size of 450 bp were prepared and quality-checked with the Agilent 2100 Bioanalyzer. Paired-end sequencing was performed on an Illumina HiSeq 2000 platform. Reads were aligned to the reference genome using HISAT2, and gene expression levels were quantified and normalized with HTSeq (FPKM > 1). Differentially expressed genes (DEGs) were identified using DESeq2 (v1.16.1) with thresholds of |logFC|  1 and p < 0.05. Bidirectional clustering was conducted using the R package pheatmap.