GSM9248719: benzo[a]pyrene, 0.133, 48 hpf, D; Danio rerio; RNA-Seq
RNA extraction was performed using the KingFisher Apex System (ThermoFisher Scientific, Waltham, MA, United States) and Quick-RNA Magbead kit (Zymo Research Corporation, Irvine, CA, United States). Samples were thawed and combined into the homogenization plate (2 mL deep 96-well plate pre-filled with 400 µL 1 mm zirconium silicate beads and 525 µL of lysis buffer) and sealed with a silicone seal. Using the Mini-BeadBeater 96 (BioSpec Products, Bartlesville, OK, United States), the plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 550µL of the supernatant was distributed over two plates, each pre-filled with 275µL of ethanol. Extraction was an automated process that using Mag beads to collect RNA, after a series of washes and treatment with DNase, the RNA is eluted in 50µL of ultra-pure water. RNA quality was assessed using the RNA ScreenTape and the 4200 TapeStation System (Agilent, Santa Clara, CA, United States). The RNA purity was assessed using the 260/230 ratio measured with the Varioskan ALF multimode plate reader (ThermoFisher Scientific, Waltham, MA, United States). RNA concentration was determined using Quant-iT broad-spectrum RNA Reagent and assay kit and Varioskan ALF multimode plate reader (standard curve R2=0.996). The four replicates of each treatment with the highest RNA concentration, which met RNA quality and purity criteria (2.3 ≥ 260/230 ≥ 1.8, RIN=10), were selected for sequencing. The resulting 80 samples were normalized using ultrapure water to the lowest concentration, 56 ng/µL. library constructuon and RNA sequencing was performed by Alithia Genomics (Epalignes, Switzerland) using their Mercurius™ BRB-SEQ service, which is a multiplexed 3' RNA sequencing library preparation technique. Samples were sequenced using NextSeq 200 with ~9.6 million reads/sample.