Gene expression analysis in untreated adult zebrafish livers (~1 year older): wild-type TAB14 compared to as3mt-/- mutants

This study was carried out to compare the changes in basal gene expression in livers of ~1 year old zebrafish adults in response to whole body mutation of the arsenite methyltransferase gene (as3mt) - 8 bp deletion in exon 3 resulting in a loss of function. The Sadler lab generated a whole body knockout of as3mt in zebrafish using CRISPR. This line a missense mutaion with a premature stopcodon (8 bp deletion in exon 3). This experiment was conducted by dissecting livers from 2 males and 2 females of WT-TAB14 (DOB: March 15, 2022) or as3mt mutant (DOB: January 19, 2022)fish. Livers were dissect under the steroscope and immediately placed in TRIzol on ice. RNA from was extracted per standard TRIzol/Chloroform method and concentrated and concentreated and DNAse treated (RNase-Free DNase Set; Qiagen) using RNeasy Mini Spin column kit (Qiagen) and resuspended in 30 uL of DNase/RNase free water (Thermo Fisher Scientifc). For library preparation, high quality of total RNA was QCed using a bioanalyzer (Agilent 2100; Agilent Technologies, Santa Clara, CA, USA). RNA was then quantified by Qubit flurometer. Next, RNA quality was measured on a Bioanalyzer (Agilent) and the only RNA with RIN score >7 were used for library preparation.mRNA library was prepared using Illumina TruSeq V2 RNA sample Prep Kit (San Diego, CA) according to the manufacturer’s protocol. Briefly, 100 ng of total mRNA was poly-A purified, fragmented, and first-strand cDNA reverse transcribed using random primers. Following second-strand cDNA synthesis, end repair, addition of a single A base, TrueSeq adapter-index was ligated to cDNA libraries, and PCR amplification of 12 cycles was done for enrichment, producing a 350-400 bp fragment including adapters. The fragment sizes and purity of the libraries were confirmed by analyzing on a Bioanalyzer 2100 (Agilent Technologies). The quantities of the libraries required for RNA-seq were determined by real-time qPCR using a KAPA library quantification kit for the NEBNext Ultra II Directional RNA Library Prep kit for Poly A. Enriched cDNA libraries were sequenced using the Illumina NextSeq550 (Illumina). Note:wttab14_female_liver2 was excluded from analyses