High-Throughput Transcriptomics Analysis of Chemical Effects on Zebrafish Liver (ZFL) and Embryonic (ZEM2S) Cells

With thousands of chemicals in commerce and the environment with incomplete ecotoxicity information, rapid identification of potential hazards is a critical need. Molecular profiling assays such as high-throughput transcriptomics (HTTr) provides an efficient means of screening chemicals for bioactivity and potential hazards in cell lines derived from ecological species. Information from HTTr screening can be used to identify concentrations of test chemicals that perturb cellular biology, as the basis for mechanistic prediction and to group chemicals based on bioactivity patterns. In this study, a set of chemicals, including drugs and chemicals found in consumer products, were tested in eight-point concentration series in two zebrafish cell lines: the ZFL liver cell line (ATCC CRL-2643) and the ZEM2S embryonic cell line (ATCC CRL-2147). At total of 42 unique chemicals were tested in each cell line. The experimental design also included vehicle control wells and reference chemicals tested in either eight-point concentration series or at a single concentration in order to assess assay performance. ZFL and ZEM2S cell lysates in 384-well format were created 24 hours following chemical exposure by adding 10 µL of 2X BioSpyder Enhanced lysis buffer to 10 µL of residual phosphate buffered saline remaining in each assay well following two rounds of washing. Lysates were created inside of a PCR hood. Plates were shipped to BioSpyder, Inc. frozen (on dry ice) using overnight shipping. Lysates were then analyzed by BioSpyder using the Zebrafish S1500+ Surrogate assay (v1.0) with standard attenuation. 2 µL of each lysate was hybridized with 2 µL of detector oligos from the assay using the following thermal cycler protocol: 10 min at 70 °C, followed by gradual decrease to 45 °C over 49 min, then incubating at 45 °C for 16-24 h. Excess oligos were then removed via nuclease digestion (90 min at 37 °C), and hybridized detector oligos were ligated (1 h at 37 °C) following respective additions of 24 µL TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured, and 10 µL of each ligation product was transferred to an amplification microplate containing 10 µL of PCR master mix per well. Ligation products were then uniquely labeled during product amplification with well coordinate-specific “barcoded” primer pairs containing universal adapters for sequencing. Samples were pooled into sequencing libraries and sequenced on an Illumina NovaSeq X Sequencing System (Illumina, San Diego, CA). The target read depth for each test sample was 1.7 x 10^6 mapped reads. *************************************************************** The table below lists SRA accessions for raw data already submitted to SRA under PRJNA1289737. ***************************************************************