Larval fathead minnows exposed for 24hr to 4-nonylphenol.
Total RNA was isolated from each homogenate using MagMAX-96 Total RNA Isolation kits (AM1830, Thermo Fisher Scientific, MA, USA) following the manufacturers instructions.  All isolated RNA was quantified with spectrophotometry (Synergy HTX plate reader and a Take3 microvolume plate, BioTek, VT, USA) and 16 randomly chosen RNA samples per 96-well plate were analyzed with electrophoresis (4200 Tapestation, Agilent, CA, USA) to confirm quality of the RNA isolation procedure (RINe > 9).   Each cDNA library was prepared from 250 ng of total RNA using commercially available kits that select for mRNA (SENSE mRNA-Seq Library Prep kit, Lexogen, NH, USA) following the manufacturers instructions.  The quality and size distribution of libraries was determined using a 4200 TapeStation, and concentrations were measured with a fluorescence-based assay (Quant-iT PicoGreen dsDNA Assay, Thermo Fisher Scientific, MA, USA).  Overall, 89 samples out of a maximum of 1152 were not submitted for RNA sequencing due to various quality control issues (ex., failed library preparation, dead or missing fish, or low-quality RNA).