RNA-Seq profiling of mESCs and their neuronal derivatives overexpressing WT DNMT1 and DNMT1Y495C mutant form
RNA-Seq profiling of mESCs and their neuronal derivatives overexpressing WT DNMT1 and DNMT1Y495C mutant form
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. The respective cell types were washed with 1X PBS and standard volume of RLT with BME was added added and scrapped to obtain the cell lysate which was then stored at -20 degree celcius till further use. Total RNA was extracted from ESC and neuron samples using Qiagen AllPrep DNA/RNA Kit according to standard instructions. RNA sequencing libraries were prepared with Illumina-compatible NEBNext® Ultra™ II Directional RNA Library Prep Kit (New England BioLabs, MA, USA) at Genotypic Technology Pvt. Ltd., Bangalore, India. 500 ng of total RNA was taken for mRNA isolation, fragmentation and priming. Fragmented and primed mRNA was further subjected to first strand synthesis followed by second strand synthesis. The double stranded cDNA was purified using NEBNext sample purification beads. Purified cDNA was end-repaired, adenylated and ligated to Illumina adapters as per NEBNext® Ultra™ II Directional RNA Library Prep protocol followed by second strand excision using USER enzyme at 37 ˚C for 15mins. Adapter ligated cDNA was purified using NEBNext beads and was subjected to 11 cycles for Indexing-(98˚C for 30 sec, cycling (98˚C for 10sec, 65˚C for 75sec) and 65˚C for 5min) and enrich the adapter-ligated fragments. Final PCR products (sequencing library) were purified with NEBNext beads, followed by library quality control check. Illumina-compatible sequencing library were quantified by Qubit fluorometer (Thermo Fisher Scientific, MA, USA) and fragment size distribution was analyzed on Agilent 2200 TapeStation.