Computational protocol: Inorganic arsenic causes fatty liver and interacts with ethanol to cause alcoholic liver disease in zebrafish

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Protocol publication

[…] Total RNA was extracted from livers dissected from 5 dpf Tg(fabp10a:nls-mCherrymss4Tg) zebrafish larvae using the Zymo Quick-RNA Micro Kit (R1050, Zymo Research) as per the manufacturer's instructions. RNA quality was determined by Agilent 2100 BioAnalyzer. RNAseq libraries were prepared according to Illumina TruSeq RNA sample preparation version 2 protocol, and library quality was analyzed on the Agilent 2100 BioAnalyzer. cDNA libraries were sequenced on the Illumina Hiseq 2500 platform to obtain 100-base-pair paired-end reads. Sequencing quality was assessed by using FASTQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/), and reads were quality trimmed using Trimmomatic () to remove low Q scores, adapter contamination and systematic sequencing errors. Reads were aligned to the Danio rerio GRCz10 reference genome assembly with Tophat 2.0.9 (). To estimate gene expression, read counts were calculated by HTSeq with ensemble annotation (; ; ). Normalization and test of differential expression using a generalized linear model were implemented in DESeq2 (; ). Adjusted P-value (FDR) <0.05 was treated as significantly different expression. We established a method to identify ‘on-off’ genes as those that were expressed in controls but not in the experimental condition, or vice versa. These were designated as genes with read counts greater than or equal to 20 in one condition and less than or equal to 5 in the other. RNAseq datasets were submitted to Gene Expression Omnibus (GEO) with the access number GSE104953. RNAseq from livers of Tg(fabp10a:nls-mCherrymss4Tg) larvae exposed to 2% ethanol from 96 to 132 hpf was previously described (; GSE56498), as were the unexposed 5 dpf controls (GSE52605; ). The data was realigned and reanalyzed for this study. […]

Pipeline specifications

Software tools FastQC, Trimmomatic, TopHat, HTSeq, DESeq2
Databases GEO
Application RNA-seq analysis
Organisms Danio rerio, Homo sapiens, Caenorhabditis elegans
Diseases Fatty Liver, Liver Diseases, Liver Diseases, Alcoholic, Drug-Related Side Effects and Adverse Reactions
Chemicals Ethanol, Arsenic, Tunicamycin