High-throughput Transcriptomics Screen of ToxCast Chemicals in HepaRG Cells
Lonza NoSpinTM HepaRG cell lysates were processed with 10 microliter 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. Lysates were then analyzed by BioSpyder using the TempO-Seq human whole transcriptome version 2 (hWTv2) assay. 2 microliter of each lysate was hybridized with 2 microliter of detector oligos from the hWTv2 assay using the following thermal cycler protocol: 10 min at 70 degrees C, followed by gradual decrease to 45 degrees C over 49 min, then incubating at 45 degrees C for 16-24 h. Excess oligos were then removed via nuclease digestion (90 min at 37 degrees C), and hybridized detector oligos were ligated (1 h at 37 degrees C) following respective additions of 24 microliter TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured, and 10 microliter of each ligation product was transferred to an amplification microplate containing 10 microliter of PCR master mix per well. Ligation products were then uniquely labeled during product amplification with well coordinate-specific "barcoded" primer pairs containing universal adaptors 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 6,000,000 total sequenced reads.