RNA-seq analysis of CD34⁺ HSPC-derived Fcells at different stages of differentiation. CD34⁺ cells from 2 independent donors were cultured, and stage-matched F cells were sorted on days 8, 11, and 14 of differentiation. (A) RT-PCR and western blot analysis of globins. RT-PCR data are normalized to mean expression of 3 housekeeping genes and shown relative to day 8 A cells as individual values and mean. (B) Principal component (PC) analysis of RNA-seq data from sorted F and A cells. Two biological replicates are shown for each time point. (C) Venn diagrams showing overlap of F-cell enriched or A-cell enriched transcripts between different days of differentiation. (D) RNA-seq data from days 8, 11, and 14 of differentiation. Average data from 2 replicates is shown. FDR <0.05; fold change >1.5. Globin transcripts and known HbF regulators (BCL11A, LRF, EIF2AK1, and SPOP) are highlighted.