Transcriptome analysis of tibial tissue

Tibial samples from the Sham, MTD, and LUT-H groups were collected for transcriptomics sequencing. Total RNA was extracted using Trizol reagent, and the concentration, purity, and integrity of the RNA were assessed using a Nanodrop 2000 spectrophotometer and agarose gel electrophoresis. The enriched mRNA was reverse-transcribed into complementary DNA (cDNA), and sequencing libraries were prepared for high-throughput RNA sequencing on an Illumina platform. Differential expression analysis was conducted using DESeq2, with genes identified as significantly differentially expressed when P<0.05 and |Fold Change|>1.5. Functional enrichment analysis, including Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotations, was performed using Goatools and KOBAS to identify biological processes, molecular functions, and metabolic pathways associated with differentially expressed genes. Data visualization was carried out using volcano plots, heatmaps, and pathway enrichment analysis to provide a comprehensive overview of the results.