High-throughput Transcriptomics Screen of 313 Reference Chemicals in U-2 OS Cells
U-2 OS cell lysates were created with 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. Lysates were then analyzed by BioSpyder using the TempO-Seq human whole transcriptome version 2 (hWTv2) assay with U-2 OS specific attenuation. 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 70 C, followed by gradual decrease to 45 C over 49 min, terminating with 45 C incubation 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, distributed across multiple lanes of a HiSeq dual flow cell and analyzed on a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA). The target depth for each test sample was 3 x 10^6 total sequenced reads.