DNA IP for 5hmC modified CpGs in DNA of ESC derived hepatocyte like cells exposed to high energy substrates.

We report the application of antibody affinity DNA immunoprecipitation followed by proton sequencing to map 5'hydroxymethylcytosine profiles in DNA of ESC derived hepatocyte like cells exposed to high energy substrates. Female H9 ESCs were maintained in mTeSR1 (Stem Cell Technologies, Cambridge UK) on matrigel (Corning, Tewksbury, USA) and differentiated into hepatocyte-like cells as previously described (Szkolnicka D, Curr Protoc Stem Cell Biol 2014;30:1G 5 1-12.). At day 20 of differentiation, cells were exposed to Sodium L-Lactate (L7022, Sigma, Gillingham, UK) 10mM, Sodium Pyruvate 1mM (Sigma, Dorset, UK) and Octanoic Acid (C2875, Sigma, Gillingham, UK) 2mM for 48 hours before harvesting. DNA was extracted using quigen DNAeasy kit. We carry out 5-hydroxymethylation DNA immunoprecipitation (hmeDIP) prior to sequencing Ion Proton P1 to report locus specific changes in 5hmC induced by LPO treatment.