GSM8374651: BPA.1_5uM; Homo sapiens; RNA-Seq
Following the exposure, cells were washed with phosphate-buffered saline (PBS) and lysed for Templated Oligo-Sequencing (TempO-Seq®) using 2xTempO-Seq lysis buffer diluted with PBS. Cell lysates were used directly for TempO-Seq library building. Gene expression changes were measured using the TempO-Seq Human Whole Transcriptome Panel v2.1 Assay, Index E, F, G (BioSpyder Technologies Inc, Carlsbad, California) following manufacturer's instructions. Briefly, 1X TempO-Seq Enhanced Lysis Buffer was added to exposed MCF-7 cells to achieve cell lysis. Cell lysates were frozen and stored at -80°C for less than six months prior to library building. At the onset of library building, thawed cell lysates and technical controls (1) Human Universal Reference RNA—uhrRNA Agilent Cat No. 740000, Santa Clara, California, and 2) Human Brain Total RNA brRNA—ThermoFisher AM7962, Waltham, Massachusetts) were added to assay plates. Freshly prepared annealing mix, containing the whole human genome Detector Oligo (DO) Pool to be annealed to the sample RNA, was added to the assay plates. The mixture was incubated at 70°C for 10 minutes, then ramped to 45°C over 50 minutes, then held at 45°C for 16 hours, and finally cooled to 25°C to hybridize the DO pool. Assay plates were frozen overnight at -20°C. Freshly prepared Nuclease Mix was added to each well of the thawed assay plates and then the assay plates were incubated for 90 minutes at 37°C to ensure removal of unbound DOs. Freshly prepared Ligation Mix was added to each well of the assay plate and then plates were incubated for 1 hour at 37°C, then immediately at 80°C for 15 minutes to ligate adjacent DOs together. For DO amplification, PCR Pre-Mix and PCR primers were mixed with the ligated assay products and then amplified on a thermocycler using the following protocol: 1) 37°C 10 minutes; 2) 95°C 1 minute; 3) 25 cycles of 95°C 10 sec, 65°C 30 sec, 68°C 30 sec; 4) 68°C 2 minutes; 5) hold at 25°C. After amplification, assay plates were stored at -20°C prior to purification. When all three assay plates in the study completed amplification, the plates were thawed and amplicons were purified using the Macherey-Nagel NucleoSpin Gel and PCR Cleanup Kit. Libraries were pooled in equimolar amounts prior to sequencing. Human TempO-Seq Whole Transcriptome - Index Sets E, F, and G were used to index libraries