GSM8969920: 2,4,5-trimethylnaphthalene, D; Danio rerio; OTHER
1mL of 1µm beads and 500µL of lysis buffer were loaded into a round bottom 2mL 96 well plate. 100µL of lysis buffer was added to tube containing pooled embryo samples and 200µL of RNA shield, the full contents of the tube were then transferred to the homogenization plate. The plate was homogenized for 1.5 minutes and centrifuged for 5 minutes at 3000 rcf. 275 µL of the supernatant was then transferred to two plates containing 275µL of ethanol. RNA extraction was performed using the KingFisher Apex system. Briefly this process uses Mag beads to collect RNA from the sample, after series of washes the RNA is eluted in 50µLof ultra-pure water. RNA quality was assessed using the Agilent 4150 TapeStation System. All samples used had RIN scores >9.8. RNA concentration was determined using ThermoFisher Quant-iT RNA Reagent and assay kit and ThermoFisher Varioskan ALF multimode plate reader in triplicate, samples with high variability between reads were repeated in triplicate. The average of the three quantification reads was used for RNA concentration normalization. Each treatment had 4-5 viable RNA extraction replicates, for treatments with 5 viable samples the 4 with the highest concentration and RIN score were used 3 control samples were used from each day group. All 94 samples were normalized to the lowest concentration sample 14.9ng/µL. 1 µL per sample of normalized RNA was used as input for library preparation following manufactures protocol for the 96-sample TempO-Seq S1500 + Zebrafish Surrogate Assay panel (BioSpyder Technologies, Inc., Carlsbad, CA, United States). Briefly, samples were added to the assay plate with an equal volume of 2x enhanced lysis buffer and processed through annealing with detector oligos, digestion, and ligation. Proper amplification was verified using real-time PCR green fluorescence. Library purification was performed using the Macherey-Nagel NucleoSpin Gel and PCR Cleanup Kit following adjustments detailed in the manufacturer's instructions. PCR amplification products were pooled into a single sequencing library 5 µL each into a 1.5mL Eppendorf tube the pooled purified libraries were sequenced using one lane of the NovaseqX 10 B flow cell (BioSpyder Technologies, Inc., Carlsbad, CA, United States).