Role of TET3 in mouse embryonic and adult hematopoiesis [E5hmC-seq]

In this study: we mapped genome-wide DNA hydroxymethylation levels in Tet3F/F (control) and cKO Tie2+/cre; Tet3F/F (cKO) Lin– bone marrow cells by enzymatic 5-hydroxymethylation sequencing (E5hmC-seq). 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 E5hmC-seq, 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 E5hmC-seq kit using 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 T4 (5hmC positive control) DNA were used as controls. Details of E5hmCseq procedure and data analyses are described in detail in the methods section of the manuscript.