GSM8779132: Hs 839.T, Phase 2, Replicate 2; Homo sapiens; RNA-Seq
For adherent cell lines cultured in multiwell plate format (6-well, 12-well, 24-well), growth media was removed and cells were gently rinsed once with a volume of 1X phosphate-buffered saline (PBS). The 1X PBS was removed and 1 mL of 1X BioSpyder lysis buffer was added to each well. Cultures were immediately inspected under a microscope to evaluate the extent of cell lysis.  Cells were then incubated for 20 minutes at room temperature protected from light. After 20 minutes, cell cultures were again inspected under a microscope to confirm efficient cell lysis. Using a P1000 pipette, the lysate contained in each well was titurated four times and used rinse the growth surface of the mutliwell plate. Lysates were then transferred to RNase-free microcentrifugre tubes and stored at -80C prior to shipment to BioSpyder for TempO-Seq whole transcriptome profiling. For cell lines cultured in suspension, the number of live cells in suspension were quantified in a small aliquote of cells using trypan blue dye exclusion. A volume of cell suspension corresponding to the desired number of cells for TempO-Seq analysis were then transfered to a sterile 1.5 mL microcentrifuge tube. Cells were pelleted at maximum speed for 2 minutes using an Eppendorf 5340R centrifuge equipped with a microtube rotor (FA-45-48-11). Residual media was removed and 1 mL of 1X BioSpyder lysis buffer was added. The cell lysates were titurated several times with a manual P1000 pipette to dissolve the cell pellet and ensure effective lysis. Lysates were stored at -80C prior to shipment to BioSpyder for TempO-Seq whole transcriptome profiling.  The TempO-Seq assay was performed on cell lysates as described (Harrill et al. 2021) without RNA extraction. 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 for Phase 1 were sequenced to a target read depth of 6.0x106 mapped reads per sample. The TempO-seq libraries for Phase 2 were generated and sequenced to a target read depth of 4.5x106 mapped reads per sample.