High-Throughput Transcriptomics (HTTr) Chemical Screening in MCF7 cells.
MCF7 cell lysates for TempO-Seq analysis were prepared as described (Harrill et al., 2021).  In brief, 6 h after chemical treatment, HTTr assay plates were removed from the incubator and media in each assay well drained to a residual volume of 10 uL.  A 10 uL volume of 2X BioSpyder lysis buffer was dispensed into each assay well. The plates were sealed with an adhesive aluminum plate cover and incubated at room temperature, protected from light, for 30 minutes. The plates were then stored at -80C prior to shipment to BioSpyder, Inc. MCF7 cell lysates were analyzed at BioSpyder, Inc. using a custom-attenuated version of the TempO-Seq human whole transcriptome version 1 (hWTv1) assay (Yeakley et al., 2017). This assay includes 21,111 probes covering 19,287 genes. ). Lysates were processed as described (House et al. 2017). In brief, 2 uL of each lysate was hybridized with 2 uL of detector oligos from the hWTv1 assay using the following thermal cycler protocol: 10 min at 70C, followed by gradual decrease to 45C over 49 min, terminating with 45C incubation for 16-24 hours. Excess oligos were then removed via nuclease digestion (90 min at 37C) and hybridized detector oligos were ligated (60 min at 37C) following respective additions of 24 uL TempO-Seq nuclease and ligation mixes. RNA/DNA duplexes were then heat-denatured and 10 uL of each ligation product was transferred to an amplification microplate containing 10 uL of PCR master mix per well. Ligation products were then uniquely labeled during product amplification (10 min at 37C, 2 min at 95C, 6 cycles of 95C for 30 s, 54C for 30 s, 72C for 120 s, 16 cycles of 95C for 30s and 72C for 120s, followed by 72C for 60 s) with well coordinate-specific barcoded primer pairs containing universal adaptors for sequencing. Samples on each plate were then pooled into sequencing libraries. Pooled sequencing libraries were then 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 3 million sequenced reads.