Novaseq 6000 mgiseq t7

Total RNA was extracted from the brain tissues of mice in the Nor, CI, and DZSM-H groups (n = 3) using TRIzol Reagent. Applied Protein Technology (Shanghai, China) constructed the cDNA libraries using RNA, and they were sequenced on an Illumina Novaseq 6000/MGISEQ-T7 instrument according to a standardized sequencing protocol. Differentially expressed genes (DEGs) between groups were analyzed using the “limma” package and determined according to the conditions as follows: |log2(Fold Change)| > 0.58 and p-value < 0.05. Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment of DEGs at both Aging-vs.-Nor and DZSM-H-vs.-Aging groups were implemented by the DAVID database (https://david.ncifcrf.gov/) and Metascape (https://metascape.org/gp/index.html#/main/step1). The protein-protein network (PPI) of reversed DEGs was constructed by STRING database (https://cn.string-db.org/), and visualized in Cytoscape 3.7.2 platform. The topological parameter of nodes and interaction edges in the network was calculated by cytoHubba plug-in, and the nodes with top degrees were considered as key targets.

RNA sequencing was performed by Applied Protein Technology (Shanghai, China). Briefly, qualified RNA samples were used for paired-end library construction using the ABclonal mRNA-seq Lib Prep Kit (ABclonal, China). PCR products were then purified (AMPure XP system). Genome sequencing was performed with an Illumina Novaseq 6000/MGISEQ-T7 instrument.