On days 0, 1 and 2, differentiating cells were incubated (5% CO2, 37 °C) with paraben concentrations ranging from 0.316 – 1000 µM and valproic acid at 600 µM for a total of 96 h, as well as the vehicle alone (0.1% DMSO).
Growth protocol
The differentiation of SBAD2 hiPSCs to neuroepithelial precursor cells was performed using the UKN1 protocol (Chambers et al. 2009) with minor changes. Briefly, hiPSCs were seeded in 1 ml pluripotent stem cell (PSC) medium (spiked with a Rho-kinase inhibitor (ROCKi)) per well on extracellular matrix protein-coated 12-well-plates at a density of 12,000 - 24,000 cells/cm² on day -3. On day 2 and day 1, the PSC medium was refreshed. On days 0, 1 and 2, medium was changed to differentiation medium, which was spiked with 21.6 µM SB431542, 0.64 µM dorsomorphin, 35 ng/ml noggin and 0.1% DMSO to induce neural differentiation. On day 4, medium was changed to a mixed medium of 75% differentiation medium / 25% N2-S and the same concentrations of SB431542, dorsomorphin and noggin as given above. On day 6, cells were collected for RNA extraction. A more detailed method description is given in the supplemental information.
Extracted molecule
total RNA
Extraction protocol
Total RNA was isolated from sonicated cell lysates with the ExtractMe Total RNA-Kit (Blirt, Poland). The concentration and purity of the RNA were determined with a NanoDrop2000 (ThermoFisher Scientific, Germany).
Label
biotin
Label protocol
The samples were amplified and labelled with biotin using GeneChip 3′ IVT Express Kit per the manufacturer’s instructions (Affymetrix, High Wycombe, UK).
Hybridization protocol
The samples were purified using magnetic beads and fragmented. 12,5 μg of fragmented RNA samples were hybridized on Affymetrix Human Genome U133 Plus 2.0 arrays (Affymetrix, Santa Clara, CA, USA). The microarray hybridization step was performed in an Affymetrix GeneChip Hybridization Oven-645 for 16 h at 45 °C and 60 rpm. Washing and staining of the hybridized arrays was completed using the GeneChip HWS Kit (Affymetrix, High Wycombe, United Kingdom) and Affymetrix GeneChip Fluidics Station-450.
Scan protocol
Stained arrays were scanned with Affymetrix Gene-Chip Scanner-3000-7G and evaluated for quality control with Affymetrix GCOS software
Data processing
All analyses were conducted using the statistical software R, version 4.2.1 Pre-processing of the data consists of the three steps background correction, normalization, and summarization, using the frozen robust multi-array average (fRMA) algorithm. This yields expression values for 54675 probe sets. The R-packages affy, frma and hgu133plus2frmavecs were used.
