To evaluate the potential effect of alcohol on the function of the 3′UTR SNPs, untransfected SK-N-BE(2) cells were grown to confluence in a 9.5 cm2 CellBIND six-well dish (Corning, Corning, NY, USA) and then treated with physiological concentrations of ethanol (10 or 20 mM) or left untreated. Following 24 and 42 h ethanol treatment, cells were harvested for RNA isolation, library preparation, and RNA sequencing. For the 42 h treatment condition, media was replaced with fresh media with or without ethanol in both ethanol treated and control cells, respectively, at 24 h. Four independent biological replicates were conducted for each condition. The mRNA was extracted from the cells using the Qiagen RNeasy kit (Qiagen). The RNA-seq samples were prepared using the TruSeq RNA Library Pre Kit v2 (Illumina) and sequenced on the Illumina HiSeq 4000 using 2 × 75 bp paired-end configuration. A GLMM model was used to identify the variants whose allelic frequencies were altered by ethanol treatment (see Supplementary Materials). The RNA-seq data is available through Gene Expression Omnibus (GEO) database (accession number: GSE131470).
