RNA-Seq Reveal Role of Bovine TORC2 in the Regulation of Adipogenesis

2.6. Construction of RNA-Seq library, quality control and sequencing After 6 days of treatment, the adipocytes total RNA was extracted (siTORC2 and siNC; n=6), The mRNA was enriched by oligo (dT) beads, and was fragmented into short fragments through fragmentation buffer, then reverse transcribed into cDNA using random primers. The second strand cDNA was generated by DNA polymerase 1, dNTP, buffer, and RNase H. The cDNA fragments were purified through QiaQuick PCR extraction kit, then end repaired. Poly (A) was added, and then ligated to Illumina sequencing adapters. Ligation products were size selected by agarose gel electrophoresis, PCR amplification, and then sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China). After filtration clean reads were obtained, which were used for subsequent analysis (Figure S2). 2.7. DEG identification The edgeR package (http://www.r-project.org/) was used for the identification of DEGs within samples. Significant DEGs were compared with a fold change of ≥1.5 and a false discovery rate < 0.05. The DEGs were subjected to Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and enrichment analysis of Gene Ontology (GO). 2.8. Functional Enrichment Analysis Online DAVID (visualization and integration discovery) software (https://david.ncifcrf.gov/) was used for functional annotation DEGs of the GO terms including biological process (BP), cellular component (CC), and molecular function (MF) enrichment analysis [23]. Additionally, KEGG Orthology based annotation system (KOBAS) software version 3.0 China (http://kobas.cbi.pku.edu.cn/) was used for KEGG pathway enrichment analysis. A P-value < 0.05 was considered as significant enrichment. Furthermore, we used Reactome pathway analysis tool for the visualization and interpretation of the annotated pathways enriched by the DEGs [24]