Bisulfite analysis of +23 enhancer methylation. (A) Methylation patterns of the Runx1 +23 enhancer in cells from in vivo–derived E14.5 MEFs, E8.5 YS CD41⁺ (E8.5 YS), E14.5 FL Lin-Sca-1⁺CD48⁻CD150⁺ (E14.5 FL), and adult marrow Lin-c-Kit⁺Sca-1⁺CD150⁺CD48⁻ (KLS + SLAM). Sequencing reactions of individual amplicons are represented by each row of circles. Open circles denote unmethylated CpGs, and solid circles represent methylated CpGs. (B) Quantification of percent CpG methylation at +23 in hematopoietic populations derived in vivo. ***P < .001; **P < .01. (C) Methylation patterns in mESC day 6 EB c-Kit⁺CD41⁺ (D6 EB) and from OP9 cocultures: GFP⁺c-Kit⁺CD45⁺ cells isolated from IRES-GFP control group at day 6 (D6 + 6 control) and HOXB4-IRES-GFP group at day 6 (D6 + 6 HoxB4) and day 11 (D6 + 11 HoxB4). (D) Quantification of percent CpG methylation at +23 in cell populations isolated during hematopoietic differentiation of mESCs. ***P < .001.