2.3 | RNA sequencing and bioinformatics
Aorta of 4-month-old male from LDLR−/− or SKI/LDLR−/− mice were homogenized in Trizol for RNA isolation using the Direct-zol™ RNA MiniPrep Kit (Zymo Research, Irvine, CA). RNA sequencing was done in Novogene Bioinformatic Technology Co. Ltd (Beijing, China). Briefly, construction of RNA sequencing library was performed on an Illumina® Hiseq platform. Paired-end clean reads were aligned to the mouse genome (GRCm38, RRID:SCR_002344), using TopHat v2.0.12. The reads numbers mapped to each gene were quantified using HTSeq v0.6.1. The expected number of fragments per kilobase of transcript sequence per millions base pairs sequenced (FPKM) of each gene was calculated based on the length of the gene and reads count mapped to this gene that reflects the relative gene expression level. Differentially expressed genes between LDLR−/− and SKI/LDLR−/− mice were analysed using DESeq R package (1.18.0). Gene set enrichment analysis was performed on differentially expressed genes (altered > 1.5 fold) to test for enrichment of specific ontologies (Subramanian et al., 2005), and the Kyoto encyclopedia of genes and genomes (http://www.genome.jp/kegg/) was used to perform pathway analysis.