Transcriptomic ac4C profiling in NAT10 knockdown acute myeloid leukemia cells

RNA modification represents an important post-transcriptional regulatory mechanism in acute myeloid leukemia (AML), however the function and mechanism of RNA acetylation ac4C in AML remains elusive. Here, we report that NAT10, as the ac4C writing enzyme, plays a critical oncogenic function in AML and represents a promising therapeutic target for AML. To understand the mechanisms underlying the function of NAT10 as an RNA ac4C writer in AML, we profiled ac4C modification in the transcriptome of MOLM13 cells using a refined ac4C RNA immunoprecipitation and high throughput sequencing (RacRIP-seq) protocol, and performed systematic calibration with a modification-free control library generated from the in vitro- transcribed MOLM13 transcriptome (referred to as IVT control) to eliminate most of the false- positive signals. We also applied the RacRIP-seq in NAT10 knockdown and control MOLM13 cells to characterize NAT10 targets. We performed a refined ac4C RNA immunoprecipitation and high throughput sequencing (RacRIP-seq) in MOLM13 cells and systematic calibration with a modification-free control library generated from the in vitro- transcribed MOLM13 transcriptome (referred to as IVT control). We also applied the RacRIP-seq in NAT10 knockdown (shNAT10-1 and shNAT10-2) and control (shNS) MOLM13 cells to characterize NAT10 targets.