title: RNA-Seq of undifferentiated HepaRG cells in culture, after 96 hours of exposure to 63257.30 nM of CDCA
design: HepaRG cells were seeded at a density of ~2000 cells per well onto collagen(I)-coated 384-well plates and kept in culture only 3 days prior to compound exposures for maintain proliferation and the undifferentiated state. Cells were seeded using Williams E medium (ThermoFisher) supplemented with plating additive (Lonza, Catalog), and 24 hours later exchanged with Williams E medium supplemented with maintenance additive (Lonza) for the duration of cultures. After the initial 3/10 days, 50 L of compound mixtures was transferred to the plates containing cells. Media was exchanged after the initial 48h of treatment. At the end of the 96-hour compound exposure period, exposure media was removed, and cells were washed once with 50 L Phosphate-buffered saline (ThermoFisher) followed by addition of 20 L of 1X TempO-Seq lysis buffer (Biospyder) to culture plates. Plates were then incubated for 15 minutes at room temperature and frozen at -80 C. TempO-Seq analysis was performed as described previously, at a targeted sequencing depth of 500 mapped read counts per transcript (Yeakley, et al., 2017).