Role of TET3 in mouse embryonic and adult hematopoiesis [EM-Seq]

In this study: we mapped genome-wide DNA methylation levels in Tet3F/F (control) and cKO Tie2+/cre; Tet3F/F (cKO) Lin– bone marrow cells by enzymatic methyl-sequencing (EM-seq). Overall design: We investigated the roles of TET3 in gene regulation in Lin– bone marrow cells. Analysis and integration of EM-seq, E5hmC-seq, and RNAseq revealed novel functions of TET3 in hematopoietic cells. For EMseq, Lin– cells were isolated from 2-month old bone marrow of either Tet3F/F and Tie2+/cre; Tet3F/F mice. DNA was extracted from Lin– cells isolated from 2 mice per genotype (Quick-DNA miniprep kit - Zymo, D3024). 200ng of DNA was used per sample per experiement. 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 EM-seq procedure and data analyses are described in detail in the methods section of the manuscript.