Ribosome profiling of HEK-293T cells stably expressing wild-type and codon-optimized coagulation factor IX.

RIBO-SEQ and mRNA-SEQ were conducted on HEK-293T cells stably expressing wild-type (WT, RefSeq NM_000133.3) and codon-optimized (CO) version of the F9 gene. The two version of the gene are drastically different in their codon usage (60.9%) but are translated into identical versions of coagulation factor IX in terms of its amino acid sequence. WT and CO (GeneArt) F9 ORFs were sub-cloned into pcDNA3.1/V5-His-TOPO (Invitrogen/Life Technologies) according to manufacturer’s instructions to generate pcDNA3.1-F9-V5-His plasmids. Each fusion construct (WTF9-V5-His and COF9-V5-His) was sub-cloned into a lentiviral vector pTK642 (gift from Dr. Kafri, University of North Carolina at Chapel Hill) at the Pacl/Sfil site. Three independent experiments have each led to the generation of RIBO-SEQ and mRNA-SEQ data for both CO and WT control.