Pcpgfree promoter lucia

Luciferase reporter assays were carried out as described previously (30 ). Sequences of the FCGR3A and FCGR3B 5′-untranslated regions were PCR amplified using primers specific for each gene (listed in Supplementary Table 1) and inserted into the CpG-free reporter vector pCpGfree-promoter lucia (Invivogen). Sequences were verified with Sanger Sequencing at the Plant-Microbe Genomics Facility (The Ohio State University). For methylation-specific assays, plasmids were methylated in vitro using M.SssI CpG methyltransferase (Thermo Fisher Scientific). Luciferase assays were carried out in HEK293T cells using TransIT-LT1 transfection reagent (Mirus Bio) according to the manufacturer’s instructions. Jurkat, YT and K562 cells were transfected with a Nucleofector device (Lonza) using Amaxa Cell Line Nucleofector Kit V with programs X-001, O-017 and T-016, respectively. Luciferase activity was assessed 48h after transfection on a DTX880 Multimode Detector (Beckman Coulter). Luciferase signals were normalized to co-transfected pGl4-CMV-firefly luciferase vector (Promega). Luciferase activity values were displayed relative to an unmodified pCpGfree-promoter lucia plasmid.