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.