title: GSM6721965: Copper sulfate bio rep 10; Pimephales promelas; RNA-Seq
library_construction_protocol: RNA was extracted from individual larvae using MagMAX™-96 Total RNA Isolation Kit (Thermo Fisher Scientific) following the manufacturer's protocol. Samples were removed from -80 °C and immediately placed on ice. MagMax lysing/binding buffer (100 µl) was added to each tube. Samples were homogenized using a Bullet Blender Storm 24 homogenizer (Next Advance, Troy, NY). RNA was quantified spectrophotometrically on a Synergy™ HTX Multi-Mode Microplate Reader using a Take3 Micro-Volume Plate (Biotek). RNA quality was assessed using a 4200 TapeStation (Agilent). Sequencing libraries were prepared using the SENSE mRNA-Seq Library Prep Kit V2 (Lexogen). Libraries were barcoded using the supplied i7 indices to allow multiplex sequencing of samples. Ten or eleven larvae (at least two from each beaker) from each treatment were used. All libraries were prepared from 250 ng total RNA according to the manufacturer's protocol supplied with the kit.