*4.13. Enhancer Testing with a Dual Glow Luciferase Assay*

HTR-8/SVneo cells were seeded at 50,000 cells/well and grown for 24 h on a 24-well plate before being transfected with *PLAGL1* or the negative control siRNA. 24 h after this, cells were transfected again using Jetprime reagent (VWR 89129-924) with a control plasmid or an enhancer cloned into the PGL4.23 vector. The following day the plates were read following the manufacturer's protocol for the Dual-glow Luciferase Assay kit (Fisher Scientific PRE2920). To determine the significance of the relative luciferase assay change, firefly luciferase values were normalized to renilla luciferase values and then the knockdown was compared to negative control values for the same replicate using a *t*-test. Experiments were run with four biological replicates for siRNA 1.
