ESCs (mESCs) were grown in ESC medium (Knockout DMEM/F12: ThermoFisher, Cat. no. 12660012; 15% ESC-grade fetal bovine serum: HiMedia, Cat. no. RM10435; 1X Glutamax: ThermoFisher, Cat. no. 35050061; 1X Antibiotic/Antimycotic: HiMedia, Cat. no. A002A; 1X Non-Essential Amino Acids: ThermoFisher, Cat. no. 11140050, 1X β-Mercaptoethanol: ThermoFisher, Cat. no. 21985023 and 1X Leukemia Inhibitory Factor: a gift from Dr. Chandrasekhar’s lab, CCMB-Hyderabad). The ESCs were plated at a density of 50,000 cells/cm2 in a tissue culture treated dish coated with 0.1% sterile gelatin (Sigma, Cat. no. G1890). After reaching confluence, the ESCs were treated with 1X Trypsin-EDTA solution (HiMedia, Cat. no. TCL034) for further passaging. ESCs were differentiated into embryoid bodies in non adherent dishes with ESC medium without LIF and then in neural stem cell media (Knockout DMEM/F12: ThermoFisher, Cat. no. 12660012 with 1X Antibiotic/Antimycotic: HiMedia, Cat. no. A002A; 20ng/ml of basic fibroblast growth factor (bFGF): ThermoFisher, Cat. no. PHG0261; 20 ng/ml epidermal growth factor (EGF): ThermoFisher, Cat. no. PHG0315; 2% StemProTM Neural Supplement: ThermoFisher, Cat. no. A1050801; 6 units/ml Heparin Solution: STEMCELL Technologies, Cat. no. 07980; 200 mM Ascorbic acid: Merck, Cat. no. A8960 and 1X Glutamax) for a week with media changes on alternate days. One day prior to differentiation, adherent tissue culture dishes were coated with 20 µg/ml laminin (ThermoFisher, Cat. no. 23017015) in DMEM high glucose medium (HiMedia, Cat. no. AL007A) with 1X Antibiotic/Antimycotic. The resultant neurospheres were plated in the laminin coated dishes in presence of NDiff-227 (Takara Bio, Cat. no. Y40002), 1X CultureOne® Supplement (ThermoFisher, Cat. no. A3320201) and 1X Antibiotic/Antimycotic media to induce neuronal differentiation. Half of the differentiation media were replenished every alternate day for a total of 10 days. At the end of 10 days, well-differentiated neurons were obtained.
Extracted molecule
genomic DNA
Extraction protocol
Genomic DNA was extracted and purified from ESC and neuron samples using Qiagen AllPrep DNA/RNA Kit according to standard instructions
Label
Cy5 and Cy3
Label protocol
Standard Illumina Protocol
Hybridization protocol
Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium mouse methylation Beadchip using standard Illumina protocol
Scan protocol
Arrays were imaged using BeadArray Reader using standard recommended Illumina scanner setting
