Genome-wide RUNX1b binding site profiling in HE. (A) Schematic representation of the generation of HE populations for DamID-seq. Runx1^ko ES cells were differentiated as EBs up to day 3.25 and sorted for FLK1 expression. FLK1⁺ cells were cultured in liquid blast conditions for 48 hours and then sorted either for cKIT⁺, TIE2⁺, CD41⁻, or FLK1⁺ to enrich for the HE population. (B) Raw DamID-seq read data from RUNX1b::Dam and control Dam samples in Runx1^ko HE. Red bars indicate the RUNX1b::Dam peaks identified by DamID-PIP showing binding to the known RUNX1 targets Sox17, AI467606, and Gfi1. (C) Motif discovery analysis on RUNX1 DamID peaks in Runx1^ko HE. Table shows the top 5 most enriched motifs. (D) Binary heat map depicting the location of RUNX1 peaks (in blue) relative to transcript coordinates. Each transcript bound by RUNX1 was divided into 5 regions: −30 kb region upstream of transcription start site (TSS), promoter (±2 kb from TSS), first intron, transcript body without the 1st intron, and +30 kb region downstream of transcript end. Numbers indicate percentage of RUNX1-bound transcripts (total number of transcripts = 7367). Only peak distribution patterns encompassing ≥ 3% of transcripts are shown. (E) Venn diagram showing overlap of RUNX1 target genes determined by RUNX1 DamID-seq in Runx1^ko HE and by RUNX1 ChIP-seq in EBP (combination of HE and hematopoietic precursors). *P = 1.84⁻⁵⁴.