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 (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. 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.