Neural differentiation of iPSCs was performed as previously described (Chambers et al. 2012; Meents et al. 2019). iPSCs were seeded as single cells at a density of 105 cells/cm2 in presence of 10 µM Y-27632. At 80-90% confluency, neural conversion was induced by dual-SMAD inhibition: For the first five days LDN-193189 (100 nM or 1 µM, Sigma-Aldrich) and SB431542 (10 µM, Miltenyi Biotec) were added to the basal culture medium consisting of knockout DMEM/F 12 containing 15% knockout serum replacement, 1 mM L-glutamine, 100 µM non-essential amino acids, 100 µM β mercaptoethanol, 100 U/mL penicillin and 100 µg/mL streptomycin (all Thermo Fisher Scientific). To accelerate neural crest specification three small molecules (3 µM CHIR99021, 10 µM DAPT, and 10 µM SU5402, all Tocris, Bristol, United Kingdom) were added between days 2 to 10. After four days, the medium was supplemented in increasing percentages with DMEM/F 12, containing 10 ml/L N2, 20 ml/L B27 minus vitamin A supplements and 100 U/mL penicillin, 100 µg/mL streptomycin (all Thermo Fisher Scientific). N2/B27 medium was added to the basal medium at 25% between days 4-5, 50% between days 6-7 and 75% between days 8-10. On day 10, cells were dissociated using Accutase and seeded on glass coverslips coated with 50 µg/mL Poly-L-Ornithine (Sigma-Aldrich) and 5 µg/mL Laminin (Thermo Fisher Scientific) and further cultured for up to three weeks with N2/B27 medium supplemented with 20 ng/mL NGF (R&D Systems, Minneapolis, Minnesota, USA), BDNF, GDNF (all PeproTech) and 200 ng/mL L ascorbic acid (Sigma-Aldrich). Medium was changed every 3-4 days.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA of iPSC-derived neural cells was harvested with the NucleoSpin Tissue kit (Macherey-Nagel, Düren, Germany).
Label
Cy5 and Cy3
Label protocol
Standard Infinium HD Methylation Assay protocol
Hybridization protocol
Bisulfite converted DNA was amplified, fragmented and hybridized to Illumina Infinium MethylationEPIC BeadChip using standard Infinium HD Methylation Assay protocol.
Scan protocol
Standard Infinium HD Methylation Assay protocol
Description
iPSC-derived neural cells after 10 days of differentiation
Data processing
Initial analysis was performed by the Genomestudio 2010.3 (Modul M Version 1.8.5). Data were normalized with internal controls according to Illumina´s standard procedures. Methylation level at each locus was calculated with the GenomeStudio Methylation module as beta-value (ranging from 0 to 1). The number of beads per feature varies between chips and beta-values were calculated as average of at least three technical replica.
