time: 7 days exposure: valproic acid concentration: 1 mM
Treatment protocol
Differentiating cell cultures were exposed for either 1 or 7 days to either 0.1, 0.33 or 1mM VPA; or 0.033 , 0.1 or 0.33 mM CBZ . VPA and CBZ were dissolved in medium and 0.25% DMSO respectively. Control samples were collected at day 0, 1, 4, 7, and 9. At day 1 and 7 also samples cultured with 0.25% DMSO were collected serving as a control for CBZ.
Growth protocol
H9 hESC were routinely cultured on mytomycin mitotically inactivated mouse embryonic fibroblast at 37˚C in hESC medium, containing DMEM-F12, supplemented with 20% Knock Out Serum Replacement (KOSR), 1mM L-Glutamine, 0.5% 5000 IU/ ml Penicillin/5000 μg/ml Streptomycin, 1% non-essential amino acids, 0.1mM β-Mercaptoethanol and 0.2µg/ml fibroblast growth factor-basic(bFGF). hESC were passaged 2-3 per week. hESC cells were differentiated as described in (Schulpen et. al 2014) . Briefly, hESC were enzymatically dissociated after incubation with Collagenase IV and transferred to bacterial culture dishes containing hESC culture medium. Within 4 days the cells formed cell aggregates, which were transferred to Poly-D-Lysine (PDL)/ Laminin coated cell culture dishes containing DMEM-F12 supplemented 1% 5000 IU/ ml Penicillin/ 5000 μg/ml Streptomycin, 1.5 mM L-Glutamine and 10% insulin, transferrin and Selenium (ITS) premix. The cell aggregates attached to the bottom of the dishes and were cultured for 3 days. At day 7 the ITS medium was replaced by neurobasal medium supplemented with 1% 5000 IU/ ml Penicillin/ 5000 μg/ml Streptomycin, N-2- and B27 premix.
Extracted molecule
total RNA
Extraction protocol
Cells ready for RNA extraction, were stored at -20˚C in RNA protect (Qiagen). Before RNA extraction, the cells in RNA protect were thawed, centrifuged and the RNA protect was discarded. The RNA was extracted using the QiaCube and RNeasy mini kit (Qiagen) including an DNase incubation step, following the manufactures manual. The extracted RNA was stored at -80˚C. The concentration of the RNA samples was measured using the Nanodrop (Thermo Scientific)
Label
biotin
Label protocol
Amplification, labeling and hybridization was performed according to Affymetrix protocols, using an automated Affymetrix Genechip console. For each individual sample 100 ng RNA was used for the biotin-labelling reaction.
Hybridization protocol
The labeled cRNA was fragmented and hybridized to the Affymetrix HT HG-U133 + PM plates.
Scan protocol
Plates were scanned with Genechip HT array plate scanner and analysed with Affymetrix HT software suite.
Data processing
Quality control and normalization of Affymetrix CEL files were performed using the ArrayAnalysis website (http://www.arrayanalysis.org/) (Maastricht University, The Netherlands) , using the Robust Multichip Average (RMA) algorithm and MBNI custom CDF version 14.
