Feline Lindhout

2 54 (Utrecht, The Netherlands). The following investigations were done in R statistical software (R Core Team, 2019) with the use of packages ggplot2 (Wickham, 2009) and pheatmap (Kolde, 2019). The raw read counts were normalized to reads per million for each gene. Genes observed in less than 5 samples were excluded from further analysis. To determine differential expression a linear regression ANOVA model was used where gene expression is explained by timepoint + biological replicate and technical replicate (nested in the biological replicate). To obtain the differences (in both p-value and effect between the timepoints) a Tukey-test was performed for each gene using the same model. We corrected for multiple-testing by pairwise comparison using the p.adjust() function in R with the ‘BH’ setting (R Core Team, 2020). Genes with an adjusted p-value < 0.05 were used for further investigation. GO enrichment was done using the hypergeometric test phyper() in R (R Core Team, 2020), the set of GO terms was obtained from Ensembl Biomart for Human genes version GRCh38.p13. Comparison between transcriptomics and proteomics was done by selecting genes present in both datasets based on their public name. Sample preparation for mass spectrometry (TMT labeling) Replicates of hiPSC-derived neurons were harvested with lysis buffer (8 M Urea, 50 mM ammonium bicarbonate (Sigma), EDTA-free protease inhibitor Cocktail (Roche)) at three distinct differential time points (days 1, 3, and 7). Lysates were sonicated on ice with a Bioruptor (Diagenode) and cleared by centrifugation at 2500 g for 10 min. The protein concentration of the samples was determined by Bradford assay. Per sample 100 µg of proteins were reduced (5 mM DTT, 55˚C, 1 hour), alkylated (10 mM Iodoacetamide, 30 min in the dark) and sequentially digested by LysC (Protein-enzyme ratio 1:50, 37˚C, 4 h) and trypsin (Protein-enzyme ratio 1:50, 37˚C, overnight). After digestion (overnight), formic acid (final concentration 3%) was used to acidify the samples and resulting peptides were afterwards desalted with Sep-Pak C18 columns (Waters). Samples were labeled with stable isotope TMT-6plex labeling, according to manufacturer’s instruction (Thermo Fisher Scientific). In short, peptides were resuspended in 80 µl of 50 mM HEPES buffer, 12.5% ACN (pH 8.5), while TMT reagents were dissolved in 50 µl anhydrous ACN. We added 25 µl of each dissolved TMT reagent to a correspondent sample according to the following scheme: day 1 (replicate A) = TMT-126 / day 1 (replicate B) = TMT-129 day 3 (replicate A) = TMT-127 / day 3 (replicate B) = TMT-130 day 7 (replicate A) = TMT-128 / day 7 (replicate B) = TMT-131 Following incubation (room temperature) for 1 hour, the reaction was quenched with 5% hydroxylamine. Differentially TMT-labeled peptides were mixed in equal ratios and dried in a vacuum concentrator. Peptide fractionation Mixed TMT-labeled peptides were solubilized in 10mM ammonium hydroxide, pH 10.0 and subsequently fractionated using basic pH reverse phase HPLC. Peptides were