RNA-Seq of zebrafish embryos (96hpf) treated with different concentrations of Triiodothyronine (T3) below acute toxicity levels against untreated control groups

The aim of this sequencing experiment was to screen for ecotoxicogenomic fingerprints for endocrine disrupting chemicals affecting the thyroid system in zebrafish (Danio rerio) embryos as aquatic vertebrate model and alternative to animal testing. Triiodothyronine (T3, CAS: 6893-02-3) was tested as a model substance for thyroidal inducing activity. In a modified version of the zebrafish embryo toxicity test (OECD 236), 15 fertilized eggs were exposed to two different sub lethal concentrations of T3 for 96 hours under semi-static conditions. Each test comprised of a low exposure (LE), high exposure (HE) and negative control (NC) group and was performed in triplicates. At 96 hours post fertilization (hpf), 10 larvae were randomly picked for each sample and pooled for RNA and protein extraction with NucleoSpin RNA/Protein kit (Macherey-Nagel). RNA quality was assessed with a 2100 Bioanalyzer system (Agilent) before coding RNA was purified (PolyA selection with TruSeq RNA Library Prep Kit v2) and sequenced on an Illumina HiSeq 4000 System (Illumina) in 50 bp single read mode, producing roughly 30 million reads per sample. Adapter sequences were removed with trimmomatic and sequences were aligned to the D.rerio reference genome GRCz11 with STAR. Counting of feature mapped reads was performed through featureCounts. Library gene count tables were then merged to a single count matrix as input for differential gene expression analysis with DESeq2.

本测序实验旨在以斑马鱼（Danio rerio）胚胎作为水生脊椎动物模型及动物实验替代模型，筛选影响甲状腺系统的内分泌干扰物的生态毒理基因组指纹。三碘甲状腺原氨酸（Triiodothyronine, T3，CAS号：6893-02-3）被选为模拟甲状腺诱导活性的模式物质。在改良版斑马鱼胚胎毒性试验（OECD 236）中，将15枚受精卵置于半静态条件下，暴露于两种不同亚致死浓度的T3溶液中，暴露时长为96小时。每组试验设置低浓度暴露组（LE）、高浓度暴露组（HE）与阴性对照组（NC），并设置三次生物学重复。受精后96小时（hpf），从每个样本中随机选取10条幼鱼并混合，使用NucleoSpin RNA/Protein试剂盒（Macherey-Nagel品牌）进行RNA与蛋白质提取。在使用TruSeq RNA文库制备试剂盒v2进行PolyA富集以纯化编码RNA之前，通过2100生物分析仪（Agilent品牌）评估RNA质量。随后在Illumina HiSeq 4000测序系统（Illumina品牌）上以50 bp单端读取模式开展测序，每个样本约产生3000万条reads。使用Trimmomatic软件去除测序接头序列，通过STAR软件将测序序列比对至斑马鱼参考基因组GRCz11。利用featureCounts软件统计比对到基因组特征的reads数目。随后将各文库的基因计数表合并为单一计数矩阵，作为DESeq2软件进行差异基因表达分析的输入文件。