title: GSM6045131: U2OS_HTTR_HTPP_COMBO_Pilot_Source_Plate_A01_6_30.000000uM_TC00000652_B22; Homo sapiens; RNA-Seq
library_construction_protocol: Twentyfour hours after chemical treatment, HTTr assay plates were removed from the incubator and media in each assay well was drained to a residual volume of 10 µL using MultiFlo FX microdispenser equipped with a vacuum-driven aspiration manifold. To create cell lysates, 10 µL volumes of 2X BioSpyder lysis buffer were dispensed into each assay well using the same MultiFlo FX instrument equipped with 1 µL peristaltic pump dispensing cassette. Plates were temporarily sealed with an adhesive film, shaken for 30 s on the stage of MultiFlow FX microplate dispenser (BioTek, Winooski, VT) and then placed in a hybridization oven (37°C) for 10 min. The adhesive films were removed, plates were re-sealed with NuncTM aluminum acrylate sealing tape and stored at -80°C. Prior to shipment, the tape was removed and reference samples (reference RNAs, bulk lysates and lysis buffer blank) (Harrill et al., 2021) were added in duplicate to each plate while keeping the plates frozen. The plates were then resealed with new NuncTM aluminum acrylate sealing tape. 384-well 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 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).