Transcriptional reporter assays

We designed oligonucleotide primers (S11 Table) with KpnI and XhoI restriction sites. Using Q5 High-Fidelity DNA Polymerase (New England BioLabs), we PCR-amplified fragments surrounding each variant from DNA of individuals homozygous for each allele and cloned the PCR product into the multiple cloning site of a minimal promoter firefly luciferase reporter vector (pGL4.23, Promega) in both orientations with respect to the reporter gene. For CDH13 signal ‘B’, rs4782722 in the 398 bp construct was altered to create missing haplotypes using the QuikChange II Site Directed Mutagenesis Kit (Agilent Technologies). Three to five isolated clones for each allele for each orientation were sequenced to verify genotype. Cells were seeded with approximately 2×10⁵ (3T3-L1) or 6×10⁵ (HeLa) cells per well in 24-well plates. Each clone was co-transfected with Renilla luciferase vector (phRL-TK, Promega) in duplicate (HeLa) or triplicate (differentiated 3T3-L1) using Lipofectamine 2000 (Life Technologies) according to manufacturer’s recommendations. After 48 hours transfection, we lysed cells with passive lysis buffer (Promega) and measured luciferase activity using the Dual-Luciferase Reporter Assay System (Promega). We normalized firefly luciferase readings to Renilla luciferase readings and normalized all readings to the average of two empty pGL4.23 vector transfections. We used 2-sided Student’s t-tests to compare allelic differences in firefly luciferase activity. All transfections were repeated independently and yielded comparable results.