RNA-Seq of larval Pimephales promelas: whole larva exposed to methidathion
Whole larvae were homogenized in Buffer RLT + beta-mercaptoethanol (Qiagen RNeasy) before transferring to 96-well plates to proceed with RNA isolation using MagMAX-96 Total RNA isolation kits (Thermo Fisher Scientific AM1830) following manufacturer protocol. RNA was quantified using a Take3 microvolume plate (BioTek, USA) and a Synergy HTX plate reader (BioTek). Quality of RNA isolation was further confirmed by testing a random set of 16 samples per 96 well plate, using an Agilent Tapestation (Agilent, Santa Clara, CA). RNA was isolated from eight individual larvae per treatment, excluding any individuals that had died during the exposure. Total RNA from individual fish (250 ng/sample) was then used to prepare sequencing libraries with SENSE mRNA-Seq Library Prep kits (Lexogen), with 15 cycles of PCR amplification. The quality and size of the libraries were checked on an Agilent TapeStation system and each pooled library for sequencing contained the full complement of samples (excluding mortalities and failed samples) from the exposure plate (up to 96 samples).