GSM7941079: TP0001651B05_4_1.000000uM_PRF.DMEM_12h_TC00283200_N20; Homo sapiens; RNA-Seq
Six hours after chemical treatment, HTTr assay plates were removed from the incubator and media in each assay well was drained to a residual volume of 10 µL using MultiFlo FX microdispenser equipped with a vacuum-driven aspiration manifold. To create cell lysates, 10 µL volumes of 2X BioSpyder lysis buffer were dispensed into each assay well using the same MultiFlo FX instrument equipped with 1 µL peristaltic pump dispensing cassette. Then, 20 µL volumes of UHRR, HBRR, BLDSMO, BLTSA and Lysis Buffer reference material were manually dispensed into duplicate wells of column 1 of each assay plate. The plates were sealed with an adhesive aluminum plate cover and incubated at room temperature, protected from light, for 30 minutes. The plates were then stored at -80°C prior to shipment to BioSpyder, Inc. Samples were analyzed by BioSpyder using a custom-attenuated version of the TempO-Seq human whole transcriptome version 1 (hWTv1) assay, which includes 21,111 probes covering 19,287 genes. Lysates were processed as described in (House et al. 2017). In brief, 2 µL of each lysate was hybridized with 2 µL of detector oligos from the hWTv1 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 hours. Excess oligos were then removed via nuclease digestion (90 min at 37°C) and hybridized detector oligos were ligated (60 min at 37°) 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 (10 min at 37°C, 2 min at 95°C, 6 cycles of 95°C for 30 s, 54°C for 30 s, 72°C for 120 s, 16 cycles of 95°C for 30s and 72°C for 120s, followed by 72°C for 60 s) with well coordinate-specific “barcoded” primer pairs containing universal adaptors for sequencing. Samples were then pooled into a series of three sequencing libraries (1 for each plate). Pooled sequencing libraries were then 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).