Pipeline publication

[…] were subsequently assessed to determine the yield, purity, and quality of the RNA using a Nanodrop (ThermoFisher) and the Agilent Technologies (Santa Clara, CA) RNA 6000 Pico Kit., For whole-transcriptome analysis, three of the RNA samples that passed quality control standards (RNA Integrity Number ≥ 7) in each treatment group (n = 12 total) were submitted to the SUNY Molecular Analysis Core for Stranded Total RNA-Seq. Libraries were prepared using standard Illumina methods and sequenced with an Illumina (San Diego, CA) NextSeq 500 instrument using 1 x 75 bp single end reads at a targeted depth ≥ 15 million (M) reads per sample. Raw FASTQ files from the sequencing were generated in Illumina BaseSpace for initial quality control (filtering out reads with a q score < 30) and alignment was performed to version mm10 of the mouse genome and annotated to RefSeq (5-2-16) using the Bowtie2 algorithm within the TopHat application., The aligned BAM files were downloaded into Partek Flow and Genomics Suite (St Louis, MO) for downstream analysis. The data summarization, quantification, and normalization of RNA counts into total reads per kilobase of gene model per million reads (RPKM) and initial single statistical analysis were performed in Partek Flow. Raw FASTQ files and normalized RPKM data are freely available in the NCBI Gene Expression Omnibus (Accession GSE100217). To identify single genes and transcripts with changes in expression, we used a two-way ANOVA to test for significant main effects of the two factors ethanol Treatment and p53 Genotype, as well as the interaction of these two factors. Whenever possible, false discovery rate (FDR) adjustment for multiple testing was performed on the main and interaction effects, with a cutoff of 0.3 or less initially used for single gene discovery in the comparisons of ethanol effects in WT mice, as well as the genotype effects and the ethanol x genotype interaction effects. Not […]

Pipeline specifications

Software tools Partek Pathway, Partek Flow, Partek Genomics Suite
Organisms Mus musculus
Diseases Mitochondrial Diseases, Fetal Diseases, Alcohol-Related Disorders, Fetal Alcohol Spectrum Disorders
Chemicals Ethanol