Assessing the Impact of Metabolic Competence on Estrogenic Chemical Bioactivity in High-Throughput Profiling Assays
VM7Luc4E2 breast cell lysates were processed with 10 L 2X Enhanced BioSpyder lysis buffer inside a PCR hood 24 hours after chemical treatment. Plates were shipped to BioSpyder, Inc. frozen (on dry ice) using overnight priority shipping. Cell lysates were then analyzed by BioSpyder using an attenuated TempO-Seq human whole transcriptome version 2 (hWTv2) assay. 2 L of each lysate was hybridized with 2 L of detector oligos from the hWTv2 assay using the following thermal cycler protocol: 10 min at 70C, followed by gradual decrease to 45C over 49 min, terminating with 45C incubation for 16-24 h. Excess oligos were then removed via nuclease digestion (90 min at 37C), and hybridized detector oligos were ligated (1 h at 37C) 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 were 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 analyzed on a NovaSeq X Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA). The TempO-seq libraries were sequenced to a target read depth of 3.0x10^6 mapped reads per sample