High-Throughput Transcriptomics Analysis of Chemicals' Effects on Zebrafish Liver (ZFL) and Embryonic (ZEM2S) Cells
ZFL and ZEM2S cell lysates in 384-well format were created 24 hours following chemical exposure by adding 10 L of 2X BioSpyder Enhanced lysis buffer to 10 L 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 L of each lysate was hybridized with 2 L 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 L TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured, and 10 L of each ligation product was transferred to an amplification microplate containing 10 L 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.