Dynamic ac4C landscape across mouse hematopoietic stem and progenitor hierarchy using the ultra-low input acRIP-seq (ULAC-seq)
Epigenetic regulation exerts a crucial role in maintaining the balance between self-renewal and differentiation of hematopoietic stem cells (HSCs), thereby sustaining normal hematopoiesis. Although emerging evidence has demonstrated the contribution of RNA N4-acetylcytidine (ac4C) modification and its catalytic enzyme Nat10 to numerous biological processes, particularly tumorigenesis, the landscape and function of ac4C in normal hematopoiesis remain unexplored. In this study, we characterize the landscape of ac4C across the hierarchy of hematopoietic stem and progenitor cells (HSPCs) using  the ultra-low input acRIP-seq (ULAC-seq) and reveal a key role of Nat10 in the maintenance of HSCs. Overall design: To further investigate the role of Nat10-mediated ac4C in HSC maintenance and differentiation, we developed an ultra-low input acRIP-seq (ULAC-seq) method, capable of handling varying amounts of input, as low as 500 ng of total RNA. We subsequently profiled ac4C modification in six distinct sorted hematopoietic populations, including HSCs, MPP, CMP, GMP, MEP, and CLP from wild-type (WT) mouse BM using ULAC-seq. We also applied ULAC-seq in Nat10 knockout BM lin- cells and control BM lin- cells to characterize Nat10 targets.