Truseq rna library pre kit v2

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).

RNA was extracted from the brain tissues using the Qiagen RNeasy kit (Qiagen, Germantown, MD, USA). The RNA-seq samples were prepared using the TruSeq RNA Library Pre Kit v2 (Illumina, Inc., San Diego, CA, USA) and sequenced on the Illumina HiSeq 2000. Paired-end libraries with an average insert size of 180 bp were obtained. Raw reads were aligned to human genome 19 (hg19) using STAR aligner version 2.5.3.a [15 (link)]. FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to evaluate RNA sequence quality. The RNA-seq data is available through the NCBI BioProject database (BLA: PRJNA551909, CE: PRJNA551908, NAC: PRJNA551775, SFC: PRJNA530758).
DNA was obtained from the same brain tissues. The Axiom Biobank Genotyping Array (Thermo Fisher Scientific, Waltham, MA, USA) was used for genotyping. Monomorphic variants, variants with call rate ≤0.98 or Hardy–Weinberg equilibrium p < 10⁻⁵, and samples with call rate <0.90 were removed using PLINK [16 (link)]. Phasing was done using SHAPEIT2 [17 (link)]. IMPUTE2 [18 (link)] was used for imputation using the 1000 Genomes Phase 1 integrated panel (excluding singleton variants) as the reference. Variants with imputation score ≥0.8 and estimated minor allele frequency (MAF) ≥0.5 were included in the analysis.