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Optimization Of A High-Throughput Transcriptomic (HTTr) Bioactivity Screen In MCF7 Cells Using Targeted RNA-Seq (SOT)
Citation:
Harrill, J., M. Martin, K. Houck, R. Judson, K. Crofton, AND R. Thomas. Optimization Of A High-Throughput Transcriptomic (HTTr) Bioactivity Screen In MCF7 Cells Using Targeted RNA-Seq (SOT). Presented at SOT annual meeting, Baltimore, MD, March 12 - 16, 2017. https://doi.org/10.23645/epacomptox.5176789
Impact/Purpose:
Poster presentation at the SOT annual meeting on Optimization Of A High-Throughput Transcriptomic (HTTr) Bioactivity Screen In MCF7 Cells Using Targeted RNA-Seq
Description:
Recent advances in targeted RNA-Seq technology allow researchers to efficiently and cost-effectively obtain whole transcriptome profiles using picograms of mRNA from human cell lysates. Low mRNA input requirements and sample multiplexing capabilities has made time- and concentration-response studies of whole transcriptome changes in human cells cultured in 384-well microtiter plate format feasible and practical as a high-throughput bioactivity screen. As proof-of-concept, human MCF7 breast adenocarcinoma cells were selected as a model system and workflows for expansion from cryogenic stocks, microtiter plate culture, dosing using an acoustic dispenser and generation of cell lysates were optimized. Cell lysates were analyzed using the TempO-SeqTM human whole transcriptome assay. To reduce sample costs and enable screening applications, attenuation of highly-expressed genes may be necessary to ensure maximal usage of available read space at a specified read depth. To determine the need for attenuation, MCF7 cells cultured in DMEM+10% HI-FBS or phenol red-free DMEM+10% CS-HI-FBS (intended to minimize estrogen receptor activation) were lysed at 30, 36 or 48 hr post-plating or, alternatively, dosed with 1 µM of an HDAC inhibitor (Trichostatin A) or vehicle (DMSO) for six hours prior to lysis. Samples were sequenced to average read depth of 3e6. The data demonstrated a wide dynamic range in raw read counts with approximately one-third of the detector oligos (DOs) registering >10 counts in each sample. In each sample, 50% of available read space was occupied by ~200-300 DOs, many of which overlapped between samples suggesting that attenuation would be a viable strategy to increase virtual read depth across multiple experimental conditions. As a follow-up, MCF7 cells cultured in either media type were treated with varying concentrations (8 points, 0.03 – 100 µM) of 44 chemicals selected from the ToxCast ph1 and ph2 libraries for either 6, 12 or 24 hours. The chemicals set spanned a broad range of biological activity and included groups of chemicals with mechanistic redundancy. The resulting data demonstrates the ability of TempO-SeqTM for use in high-throughput chemical screening. The data was used to develop HTTr analysis workflows and to inform the design of larger chemical screening studies. This abstract does not necessarily reflect USEPA policy.
URLs/Downloads:
DOI: Optimization Of A High-Throughput Transcriptomic (HTTr) Bioactivity Screen In MCF7 Cells Using Targeted RNA-Seq (SOT)