GSM8404938: valproic acid_10_24h_1pool1_S51; Homo sapiens; OTHER
At the 24h and 10d time points media were removed, and spheroids were washed with Dulbecco's phosphate-buffered saline (DPBS; Thermo Fisher Scientific Waltham, MA). Spheroids were lysed by adding 10 µl of 1X TempO-Seq lysis buffer (BioSpyder Technologies Inc Carlsbad, CA) to each well. To ensure complete lysis, spheroids were incubated for approximately 10 minutes at 37°C, pipetted up and down several times, and then frozen at -80 °C until library building. Lysates and technical RNA were hybridized to the detector oligo mix following the manufacturer's instructions provided in the TEMPO-SEQ Human Tox + Surrogate with Standard Attenuation Transcriptome Kit (96 samples). This was followed by nuclease digestion to eliminate excess oligos, detector oligo ligation, and subsequent amplification of correctly ligated oligos, all performed in accordance with the manufacturer's guidelines. During the amplification stage, each sample was linked to a sample-specific barcode, enabling the pooling of samples for sequencing purposes. Pooled samples were purified using NucleoSpin Gel and PCR clean-up kits from Takara Bio USA, Inc. (Mountain View, CA). The libraries were sequenced in-house using a NextSeq 500 High-Throughput Sequencing System (Illumina, San Diego, California), employing a 50-cycle protocol from a 75-cycle high-throughput flow cell.