title: RNA-seq of IGF-1R in atherosclerosis
design: RNA-seq of IGF-1R in atherosclerosis
library_construction_protocol: We generated mice with endothelial-restricted over-expression of human wildtype IGF-1R (hIGFREO/ApoE-/-) or a signaling defective K1003R mutant human IGF-1R (mIGFREO/ApoE-/-) Total RNA was prepared from tissues using TriZol reagent (Sigma). RNA sequencing was performed by sequencing facilities at the University of Leeds. After the QC procedures, mRNA from eukaryotic organisms is enriched using oligo(dT) beads. First, the mRNA is fragmented randomly by adding fragmentation buffer, then the cDNA is synthesized by using mRNA template and random hexamers primer, after which a custom second-strand synthesis buffer (Illumina) , dNTPs, RNase H and DNA polymerase I are added to initiate the second-strand synthesis. Second, after a series of terminal repair, A ligation and sequencing adaptor ligation, the double-stranded cDNA library is completed through size selection and PCR enrichment.