Trichostatin A

TempO-Seq reagents are commercially available from BioSpyder Technologies, Inc. These are proprietary, and consist of a 2X Lysis Buffer for making cell lysates or to complement purified RNAs, a DO Pool for measurement of targeted RNAs, Annealing, Nuclease and Ligation reagents, Amplification reagents for use with custom dual indexed adapters for Illumina sequencing instruments, and a custom Index 1 sequencing primer. Other reagents (PBS, ethanol, TE, purified water) were obtained from VWR or Sigma Aldrich. Libraries were purified using the NucleoSpin Gel and PCR Clean-up kit (Macherey-Nagel cat # 740609.50). Sequencing was performed by service providers using Illumina MiSeq, NextSeq 500 or HiSeq 2500 instruments. Sequencing quality was equivalent across these instruments.
Total RNAs for TempO-Seq assay testing included the MAQC Universal Human Reference RNA (Agilent, cat#740000), MAQC Human Brain Reference RNA, and a selection of human tissue total RNAs (Thermo Fisher Scientific, cat # AM6050 and others). Synthetic RNAs for testing absolute sensitivity and fold change were the ERCC ExFold RNA Spike-In Mixes, obtained from Thermo Fisher Scientific (cat # 4456739). For the RNA-Seq cross-platform comparison, MCF-7 and MDA MB 231 cells were lysed according to the TempO-Seq protocol and RNA was purified using TRIzol (Thermo Fisher cat # 15596026). Total RNAs were assayed in both the whole transcriptome TempO-Seq assay (12 replicates each) and in RNA-Seq (6 replicates each), using ribosomal RNA depletion (KAPA Stranded RNA-Seq with RiboErase kit, Kapa cat # KK8483).
Cell lysates were made from MCF-7 or MDA MB 231 cells grown in DMEM with 10% FBS, washed once with 1X PBS before lysis. Cells were lysed at ~2,000 cells/μL in 1X TempO-Seq Lysis Buffer in PBS by incubation for 10 minutes at room temperature, followed by storage at -80°C. Trichostatin A (TSA) was sourced from Sigma Aldrich (cat # T8852) and dissolved in DMSO for the compound treatment titration, ranging from 10 nM to 100 μM TSA in 0.1% DMSO in 3-fold increments. For profiling TSA responses across cell types, MCF-7, PC-3, and HL-60 cells (undifferentiated or differentiated for 5 days with DMSO or with all-trans retinoic acid), were exposed to 1 μM TSA in 0.1% DMSO or DMSO alone for 6 hours before washing with PBS, cell lysis and storage at -80°C.

A more detailed description of materials and methods used can be found in File S1.
Briefly, the expression stability of 22 widely used RG (Table S2 in File S1) was investigated across a total of 32 experimental settings with hepatocyte-like cell types, including freshly isolated primary human hepatocytes [5] (link) at defined time points in cell culture (subgroup “primary hepatocytes”, PH), and HepG2 and Huh-7.5 cells treated with Chloroquine, Actinomycin D (ActD) [6] (link), Trichostatin A [7] (link) and DMSO - commonly used drugs with significantly differing effects in tissue culture - for different durations without passaging (subgroup “drug and density”, DD), or cultured for 14 days under a variety of conditions altering cell maturity status (subgroup “culture conditions”, CC) [8] (link), [9] (link) (all experimental settings listed in Table S1 in File S1). After RNA isolation and RT-qPCR, individual data sets of the samples, each containing Cycle Threshold (CT) values for all reference genes (primer details in Table S2 in File S1) and some exemplary genes of interest (target genes, TG; Table S3 in File S1) were further analysed in silico.
Similar to previous examinations of non-hepatic cell types [2] (link), [3] (link), the geNorm [10] (link), Bestkeeper [11] (link), and Normfinder [4] (link) algorithms were used to evaluate and rank candidate RG.

As Trichostatin A and Vorinostat possess the best binding affinities towards the RFC4 protein active site, these two complexes were chosen to carry out a 100 ns molecular dynamics simulation (MD). GROMACS-2022 was manipulated for this simulation, where the CHARMM36 force field was selected for RFC4 protein topology preparation.
Similarly, CHARMM FF via the general force field (CGenFF) server was utilized in preparing the topology of Trichostatin A and Vorinostat molecules. A dodecahedral box was used for solvating the complexes with 10 Å boundary conditions, and ions were added employing the steepest descent minimization algorithm to neutralize the complex. Subsequently, the same algorithm minimized the system’s energy with a 10.0 kJ/mol cut-off. Afterward, the systems were subjected to both NVT and NPT equilibration processes for 10 ps with a time step of 2 fs. Finally, the obtained complexes were subjected to a 100 ns MD simulation. The Molecular Mechanics Poisson Boltzmann Surface Area (MM/PBSA) [80 (link)] was deployed for binding free energy calculations, and the solvent-accessible surface area (SASA) model was applied for non-polar solvation energy calculations [81 (link)].

FOXE1-silenced cell lines were cultured in a 10-cm dish with 1 × 10⁶ cells each. After overnight culturing, the cells were treated with 5-aza (10 μmol/L) for 72 h and TSA (300 nmol/L) for 24 h. Finally, we harvested the treated cells and extracted DNA and RNA for use.