TNFRSF1A minigene constructs were made using a ~1.5 kb genomic region comprising rs1800693 and spanning the region from introns 5 to 8. Site-directed mutagenesis was performed using the QuikChange II XL Site-Directed Mutagenesis Kit (Stratagene). RNA was isolated from HEK 293T cells (ATCC) using the RNeasy Micro Kit (Qiagen) and cDNA was synthesized using the High Capacity RNA-to-cDNA Kit (Applied Biosystems), according to the manufacturer’s instructions. Minigene splicing was analyzed by PCR amplification of cDNA using primers specific to the SD and SA sites. All resulting amplification products were sequenced.
