Investigating RXR dependent changes in mRNA expression by using novel chemical modulators
Samples were collected by aspirating stimulation medium and washing each well with PBS once. Thereafter cells were lysed using the lysis buffer from  E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer's instructions. HEK293T cells were seeded at a density of 3 · 106 cells per well in 12-well plates and complete culture medium. After 24 h, medium was changed to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS for synchronization over 24 h. Medium was changed again to DMEM high glucose supplemented with sodium pyruvate (1 mM), penicillin-streptomycin (100 U/mL and 100 µg/mL, respectively) and 0.2% FCS, additionally containing either test compounds  (1 µM) in 0.1% DMSO or 0.1% DMSO. After 17 h, cells were harvested the cells.  Total RNA was isolated using E.Z.N.A. Total RNA Kit I (Omega Bio-tek) according to the manufacturer's instructions. RNA concentration and purity were assessed using a NanoDrop One UV–vis spectrophotometer (Thermo Fisher Scientific) at 260/280 nm. Novoegene repeated the isolation using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. For the directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, USER enzyme digestion, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.