title: TempO-Seq of larval Pimephales promelas whole body after 24 h exposure to PFTP (DTXSID70379295)
design: At the end of each exposure period, each well was inspected under a dissecting microscope. Mortalities were enumerated and any dead animals removed.  Volume in each well was removed and replaced with 60 l of homogenization buffer (Biospyder) and a 2.3 mm stainless steel bead added. The fish were then homogenized in 96 well plate format using a bead mixer mill (Omni-Bead Ruptor; 30 Hz for 2 min). Homogenates corresponding to the same well position on each of the three replicate plates (all the same treatment) were combined into a pool consisting of homogenate from three individual fish (except in the case of mortality on one or more plates). This resulted in five replicate pools (independent biological replicates) per treatment, each representing the mean of three individuals. For controls there were eight replicate pools, each representing the mean of three individuals, however, the controls were shared by two test chemicals (PFAS) tested on the same plate. Pooled homogenates were stored at -80C until shipped to Biospyder Technologies Inc. (Carlsbad, CA) for analysis using a custom fathead minnow TempO-Seq (Yeakley et al. 2017) assay protocol. TempO-Seq libraries were prepared by ligating detector oligos to corresponding transcripts present in the whole-body homogenates. Ligated transcripts were subjected to PCR amplification which also introduces adaptors for sequencing and a sample-specific barcode. Resulting sample specific TempO-Seq libraries were subsequently pooled together and sequenced on an Illumina sequencer (NextSeq 500). Sequence reads were then demultiplexed based on sample-specific bar codes and a FASTQ file was generated for each sample.