eQTL mapping and TWAS are widely used to contextualize GWAS, yet the impact of RNA-seq processing choices remains unexplored. We find that RNA-seq quantification method and transcriptomic reference substantially affect eQTL detection and gene expression prediction with significant downstream impact on colocalization and TWAS results. Our findings demonstrate that seemingly minor methodological decisions substantially affect these common analyses, highlighting the need for standardized practices to ensure reproducible genetic association studies.