RNA-seq of immortalized bovine myoblast
Primary cells (passage 3) and immortalized cells (passage 50) were compared to evaluate the impact of immortalization and extended culture on myogenic and adipogenic lineages. Total RNA was extracted using using an RNeasy Mini Kit (Qiagen, Hilden, Germany). cDNA libraries were prepared using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, USA), and sequencing was performed on the Illumina NovaSeq X platform to generate paired-end reads (2  101 bp). Raw sequencing reads were processed with Trimmomatic v0.38 for adapter and quality trimming, aligned to the Bos taurus reference genome (ARS-UCD2.0) using HISAT2 v2.1.0, and summarized at the gene level using featureCounts based on Ensembl release 115 gene annotation. Normalization was performed using the trimmed mean of M-values (TMM) method in the edgeR package, and expression values were calculated as log2 counts per million (logCPM). Genes with zero variance were excluded prior to principal component analysis (PCA), which was performed using the prcomp function with scaling. Multidimensional scaling (MDS) analysis was conducted using the limma package. Because only technical replicates were available, statistical inference for differential expression analysis was not performed. Instead, gene set enrichment analysis (GSEA) was conducted using ranked gene lists based on log2 fold-change values calculated from mean CPM values with a pseudocount of 1. Bovine genes were mapped to human orthologs using the biomaRt package. Genes without valid human gene symbols were excluded, and when multiple bovine genes mapped to the same human gene symbol, the gene with the highest absolute fold-change was retained. GSEA was performed using the fgsea package with Hallmark gene sets (MSigDB category H, Homo sapiens) obtained from the msigdbr database, using the fgseaMultilevel algorithm with minSize = 15 and maxSize = 500. Pathways with an adjusted p-value (padj) < 0.05 were considered significantly enriched.
SUB16241201
MyoTERT_2