ONP Methylation of LUHMES Neurons
LUHMES passage 4 containing 5 million cells was flash frozen. Two ml of lysis buffer containing 100 mM NaCl, 10 mM Tris-HCl pH 8.0, 25 mM EDTA, 0.5% (w/v) SDS and 100g/ml Proteinase K was added to cell pellet. Reaction was incubated at RT until the solution was homogenous. The lysate was then treated with 20g/ml RNase A at 370C for 30 minutes and cleaned with equal volumes of phenol/chloroform using phase lock gels (Quantabio Cat # 2302830). The DNA was precipitated by adding 0.4X volume of 5M ammonium acetate and 3X volume of ice-cold ethanol. The DNA pellet was washed twice with 70% ethanol and resuspended in an elution buffer (10mM Tris, pH 8.0). Purity of gDNA was accessed using NanoDrop ND-1000 spectrophotometer. DNA was quantified with Qbit 2.0 Fluorometer (Thermo Fisher Scientific, MA). Integrity of the HMW gDNA was verified on a Femto pulse system (Agilent Technologies, Santa Clara, CA) where majority of the DNA was observed in fragments above 100 Kb.  Sequencing libraries were prepared from 1.5g of high molecular weight gDNA using the ligation sequencing kit SQK-LSK114 following instructions of the manufacturer with the exception of extended incubation times for DNA damage repair, end repair, ligation and bead elutions.