Establishment of RUNX1b-DamID for mammalian cells. (A) Schematic representation of the DamID technique. RUNX1b binding sites are marked by methylation of nearby GATC sites. Binding sites are isolated from genomic DNA using the methylation-specific restriction enzyme DpnI, amplified by PCR and subjected to high throughput sequencing. Sequencing data were analyzed by DamID-seq analysis pipeline, DamID-PIP. (B) Csf1r luciferase assay in HEK293T cells with Runx1b, Runx1b::dam, and untethered dam cotransfected with Cbfβ. *P < .05 (n = 4). (C) EHT assay in the iRunx1b::dam^Runx1−/− and idam^Runx1−/− mouse ES lines. HE cells were cultured for 3 days in the presence or absence of dox. (Left) Representative CD41/GFP FACS plots. GFP marks Dam transgene expression. (Right) Frequency of CD41⁺ cells. *P < .05 (n = 4). (D) (Left) Representative images of iRunx1b::dam^Runx1−/− and idam^Runx1−/− aggregates at day 13 of culture in the presence or absence of dox. Dox-treated iRunx1b::dam^Runx1−/− aggregates generate round hematopoietic cells marked by red arrows. (Right) Frequency of CD45⁺ cells in iRunx1b::dam^Runx1−/− and idam^Runx1−/− aggregates at day 13 (FACS). ns, not significant.