吉林大学

Total RNA was extracted from both NDNs and LDNs using TRIzol reagent, in accordance with the manufacturer’s instructions (Trans Gen, Beijing, China). RNA concentration and quality were assessed using a microplate reader (Synergy HTX, BioTek). RNA samples were then submitted to BGI Service Co., Ltd. (service ID #F23A040014029, Wuhan, China) for RNA sequencing and subsequent bioinformatics analysis. Raw sequencing data were filtered using SOAPnuke software (v1.5.6) to obtain clean reads. These clean reads were aligned to the reference genome using HISAT2 software (v2.1.0). The Dr. Tom System from BGI (https://biosys.bgi.com) was used for data analysis, plotting, and mining. DEGs were identified using FDR-adjusted q-values < 0.05 and fold changes > 2. To elucidate the functional implications of DEGs, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analyses were performed with the Dr. Tom System; results were visualized using a bubble diagram. GSEA was performed using KEGG gene sets, with significance defined as a NES ≥ 1.0, a nominal p-value < 0.05, and an FDR-adjusted q-value ≤ 0.25.