Pgl4.11b luc2 vector

To construct a reporter plasmid containing the MERTK promoter region, this 1,625-bp region in MERTK was amplified from genomic DNA samples, using the NM_006343.2 reference sequence and primers that contained recognition sites for the HindIII and XhoI restriction endonucleases. The amplified products were inserted into the pGL4.11b[luc2] vector (Promega Corporation, Fitchburg, WI, USA). To construct a plasmid containing the wild-type MERTK gene, a vector (Addgene plasmid 23900) was purchased (Addgene, Cambridge, MA, USA)24 (link) and subcloned into the pcDNA3.1 (+) vector (Life Technologies Corporation). Genetic variations in the promoter and in the coding region were obtained using the QuikChange^® II Site-Directed Mutagenesis Kit (Agilent Technologies, Santa Clara, CA, USA). All DNA sequences were confirmed by direct sequencing. The primers used in this study are listed in Supplementary Table 1.

To construct the ST3GAL5 reporter plasmid, 2040-bp regions of the gene were amplified and inserted into the pGL4.11b[luc2] vector (Promega Corporation, Fitchburg, WI, USA). The ST8SIA1 reporter plasmid was constructed by amplifying and inserting 1977-bp regions into the pGL4.11[luc2P] vector (Promega Corporation). To evaluate the effect of variants in the coding region on ST3GAL5 expression, we constructed the ST3GAL5 reference plasmid; ST3GAL5 cDNA was purchased (GE Healthcare Bio-Sciences, Pittsburgh, PA, USA) and subcloned into the pcDNA3.1(+) vector (Life Technologies Corporation, Carlsbad, CA, USA). Haplotypes or variants in the promoter region and a nonsynonymous variant were generated using a QuikChange2® II site-directed mutagenesis kit (Agilent Technologies, Santa Clara, CA, USA). All DNA sequences were confirmed by direct sequencing. Supplementary Table 1 (only online) lists the primers used in this study.