Roles of DNA hydroxymethylation versus DNA formylation and carboxylation in neural stem cells [RNA-seq]

TET enzymes are highly expressed in neural stem cells (NSCs). They oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) in a step-wise manner. In this study we have generated NSCs that only lack the Tet formylation and carboxylation activities (Tet-TFoCa) and have compared their multipotency, transcriptome and epigenome to NSCs that lacked all three Tet activities (Tet-TMut). Our findings reveal unique roles for DNA hydroxymethylation vs DNA formylation and carboxylation in multipotency of NSCs. RNA was extracted from neural stem cells at passage one from three lines of wild type and 2 lines of Tet-FoCa and 2 lines of Tet-TMut NSCs using Omega EZNA Total RNA kit. Library preparation and RNAseq were performed by Novogene (https://en.novogene.com/) on a Illumina Novoseq 6000 platform and 20-30 million reads were generated per sample. Details of RNAseq procedure and data analyses are described in detail in the methods section of the manuscript.