Gene expression analysis in untreated or 1 mM iAs (6-120 hours post fertilization; hpf), pooled (10) whole larvae 5 days post fertilization (dpf): wild-type TAB14 compared to as3mt mutant larvae [whole lavae]

This study was carried out to compare the changes in gene expression in whole larvae 120 hours post fertilization zebrafish larvae in response to whole body mutation of the arsenic methylation gene (as3mt) - 8 bp deletion in exon 3 with and without 6-120 hpf exposure to 1 mM sodium arsenite (iAs). The Sadler lab generated a whole body mutation 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 natural spawnings between an incross of F2 homozgyous as3mt knockouts in an WT-ABNYU background in comparsion with wild-types (WT-TAB14). Mating tanks were set up at approximately 4:30 PM the night before and embryos were collected at 10:30 AM the following morning. Following collection, embryos were divided into 10 mL wells, 20 embryos per well in embryo medium (Zebrafish Information Network). 1 mM iAs was added to treatment groups at 6 hpf. At approximately 119 hpf, pools of 10 whole larvae were collected for each condition (WT or as3mt mutant and 0 or 1 mM iAs: four groups) and immediately placed into 500 uL of TRIzol (Thermo Fisher, 15596026). RNA from was extracted per standard TRIzol/Chloroform method 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. 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).