GSM8865227: Met_bestradiol_E2758_1_0.0001uM_TC00003602_G12; Homo sapiens; RNA-Seq
At 24 hours after treatment with condition media, assay plates were removed from the incubator, assay wells were washed twice with 1X phosphate-buffered saline and the final PBS wash was aspirated from each well leaving a residual volume of 10 µL.  Cells were subsequently lysed by adding 10 µL of 2X BioSpyder Enhanced Lysis buffer.  Assay plates were then sealed, agitated for 30 seconds on the stage of a MultiFlo FX washer / dispenser and then placed in a hybridization oven at 37°C for 10 minutes. Plates were then immediately transferred to a -80°C freezer for storage. Plates were subsequently shipped frozen to BioSpyder Technologies (Carlsbad, CA) for analysis with the human whole transcriptome TempO-Seq assay.  The TempO-Seq assay was conducted directly on cell lysates with no RNA extraction step. Plates containing cell lysates were shipped to BioSpyder, Inc. frozen (on dry ice) using overnight priority shipping. Cell lysates were then analyzed by BioSpyder using the unattenuated TempO-Seq human whole transcriptome version 2 (hWTv2) assay, which includes 22,537 probes covering 19,703 genes. 2 µL of each lysate was hybridized with 2 µL 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 µL TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured, and 10 µL of each ligation product were transferred to an amplification microplate containing 10 µL 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 TempO-seq libraries were sequenced to a target read depth of 3.0x106 mapped reads per sample.