title: GSM2816951: VEHICLE_24h_rep18; Homo sapiens; OTHER
library_construction_protocol: RNA was isolated from cells using TRIzol reagent (Invitrogen) and further purified by RNeasy MinElute Cleanup (Qiagen).Differential gene expression patterns and  concentration-response relationships were analyzed using TempO-Seq™ (BioSpyder Technologies, Inc.,  Carlsbad, CA) (Yeakley et al. 2017), a targeted RNA sequencing technology 119 focused on a surrogate  transcriptome panel comprising 2982 transcripts, as described previously (Grimm et al. 2016). The  sequencing library was prepared according to the manufacturer's guidelines and as previously described  (Grimm et al. 2015). In brief, RNA in 2μl of each cell lysate were hybridized with the provided detector  oligo pool mix (2μl per sample) using the following thermocycler settings: 10 min at 70°C, followed by  gradual decrease to 45°C over 49 min, and ending with 45°C for 1 min. Subsequent steps included  nuclease digestion (90 min at 37°C) ligation step (60 min at 37°C, followed by heat denaturation at 80°C  for 30 min) following addition of 24μl nuclease mix and 24μl ligation mix. 10μl of each ligation product  were then transferred to a 96-well amplification microplate containing 10μl of PCR mix per well. The  ligation products were then uniquely labeled during product amplification, when well-specific,  “barcoded” primer pairs were introduced to templates. Sequence-based barcoding is an essential step  allowing for correct identification and recognition of transcript-specific sequencing counts. 5μl of sample  amplicons from each well were subsequently pooled into a single sequencing library. The TempO-Seq  library was further processed using a PCR clean-up kit (Clontech, Mountain View, CA) prior to  sequencing at Texas A&M University Genomics & Bioinformatics Services. Sequencing was achieved  using a 50 single-end read mode in a rapid flow cell (2 sequencing lanes for increased sequencing depth)  on a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA). Sequencing  readouts were demultiplexed to generate FASTQ files, and passed all internal quality controls. Differential gene expression patterns and concentration-response relationships were analyzed using TempO-Seq™ (BioSpyder Technologies, Inc., Carlsbad, CA) (Yeakley, Shepard et al. 2017), a targeted RNA sequencing technology focused on a surrogate transcriptome panel comprising 2982 transcripts, as described previously (Biopyder toxpanel library DO-01-096) (Grimm, Iwata et al. 2016). The sequencing library was prepared according to the manufacturer's guidelines and as previously described (Grimm, Iwata et al. 2015). In brief, RNA in 2µl of each cell lysate were hybridized with the provided detector oligo pool mix (2µl per sample) using the following thermocycler settings: 10 min at 70°C, followed by gradual decrease to 45°C over 49 min, and ending with 45°C for 1 min. Subsequent steps included nuclease digestion (90 min at 37°C) ligation step (60 min at 37°C, followed by heat denaturation at 80°C for 30 min) following addition of 24µl nuclease mix and 24µl ligation mix. 10µl of each ligation product were then transferred to a 96-well amplification microplate containing 10µl of PCR mix per well. The ligation products were then uniquely labeled during product amplification, when well-specific, “barcoded” primer pairs were introduced to templates. Sequence-based barcoding is an essential step allowing for correct identification and recognition of transcript-specific sequencing counts. 5µl of sample amplicons from each well were subsequently pooled into a single sequencing library. The TempO-Seq library was further processed using a PCR clean-up kit (Clontech, Mountain View, CA) prior to sequencing at Texas A&M University Genomics & Bioinformatics Services. Sequencing was achieved using a 50 single-end read mode in a rapid flow cell (2 sequencing lanes for increased sequencing depth; mean reads per gene = 212) on a HiSeq 2500 Ultra-High-Throughput Sequencing System (Illumina, San Diego, CA). The high-expression genes listed in Supplementary Table 1 were attenuated to allow for more sequencing depth. Sequence cluster identification, quality pre-filtering, base calling and uncertainty assessment were conducted in real time using Illumina's HCS 2.2.68 and RTA 1.18.66.3 software with default parameter settings. Sequencing readouts were demultiplexed to generate FASTQ files, and passed all internal quality controls.
attributes.tag: GEO Accession
attributes.value: GSM2816951