Roles of DNA hydroxymethylation versus DNA formylation and carboxylation in neural stem cells [EM-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. DNA was extracted from neural stem cells at passage one from one line of each genotype (Zymo Quick-DNA Miniprep kit). 200 ng of DNA was used per sample. Libraries were prepared following NEBNext EM-seq kit following manufacturer instructions and subjected to 300-bp paired-end sequencing at Novogene using their NovaSeq X Plus (PE150-25B) platform. Spike-in lambda (negative control) and pUC19 (5mC positive control) DNA were used as controls. Details of EMseq procedure and data analyses are described in detail in the methods section of the manuscript.