GSM5984788: BEAS-2B_DTXSID8020250_4_5ppm [TC00504137_E04]; Homo sapiens; RNA-Seq
An optimized whole cell lysis procedure was used to preserve the RNA of both BEAS-2B and pHBEC samples. After a 4 h recovery post 2 h test agent exposure, the two technical replicate inserts from each exposure condition were placed on a new 24-well plate (Corning, NY). A single replicate insert of the incubator control cells was also preserved for whole cell lysis. Prior to adding the lysis buffer, the cells were thoroughly washed twice with DPBS to remove any remaining apical mucus or surfactant which may interfere with complete lysis. When transferring the BEAS-2B cells or pHBECs, the BioSpyder lysis buffer (Carlsbad, CA) was added to each of the samples and allowed to incubate for 15 minutes at room temperature. Each sample was mixed and pipetted into an individual well in the 384-well optical imaging plate (Corning, NY). After placing each of the samples into the 384-well plate, it was sealed with ThermalSeal RT 2mil Film (Sigma-Aldrich, MO) and stored in a -80°C freezer until shipment to BioSpyder for analysis. 384-well plates containing cell lysates were shipped to BioSpyder, Inc. frozen (on dry ice) using overnight priority shipping. BEAS-2B and pHBEC lysates were then analyzed by BioSpyder using the TempO-Seq human whole transcriptome version 2 (hWTv2) assay, which includes 22,537 probes covering 19,703 genes. Lysates were processed as described in. In brief, 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 target depth for each test sample was 6 x 10^6 total sequenced reads.