Del2Rec clonal HUDEP-2 cell lines give insight into the HBG reactivation mechanism. (A) The RT-qPCR analysis of HBG mRNA levels in proliferating (left, steel blue bars) and 10-day differentiated (right, gray bars) clonal cell lines. Expression was normalized to actin mRNA and fold-change was calculated over proliferating control cells. (B) Same as in panel A but fold-change of HBB mRNA. (C) RT-qPCR analysis of the HBB, HBG1, and HBG2 mRNA levels in proliferating clonal cell lines. HBB, HBG1, and HBG2 mRNA expression was normalized to actin and displayed as a percentage of HBB + HBG1 + HBG2. (D) Same as panel C but the cells were differentiated for 10 days. (E) Schematic representation of the primers used in the RT-qPCR analysis of the HBG transcripts (top). The fold-change, normalized for primer efficiency based on DNA amplification with HBG2 exon 2 set to 1, shown in arbitrary units for a ΔBCL11A clone and 2 Del2Rec deletion clones (bottom). (F) HbF of 2 clonal Del2Rec deletion cell lines, as measured by HPLC. The percentage of HbF calculated over the total Hb tetramers. (G) Representative flow cytometry plots showing the percentage of HbF positive cells in proliferating clonal cell lines (top row) and after 10 days of differentiation (bottom row). (H) The ATAC-seq tracks of the HBB gene cluster in differentiated (10 days) WT HUDEP-2 cells and 2 Del2Rec clones. The horizontal dashed lines indicate the deletion generated in Del2Rec clones. The signal was scaled to the genome-wide TSS signal. Max scales were set to the highest scaled signal in the HBB locus. (I) H3K4me3 CUT&RUN tracks of the HBB gene cluster in 2 differentiated WT HUDEP-2 samples and 2 Del2Rec clones. The horizontal dashed lines indicate the deletion generated in Del2Rec clones. Scaling is based on the average coverage at the HBA locus. WT, wild-type.

Del2Rec clonal HUDEP-2 cell lines give insight into the HBG reactivation mechanism. (A) The RT-qPCR analysis of HBG mRNA levels in proliferating (left, steel blue bars) and 10-day differentiated (right, gray bars) clonal cell lines. Expression was normalized to actin mRNA and fold-change was calculated over proliferating control cells. (B) Same as in panel A but fold-change of HBB mRNA. (C) RT-qPCR analysis of the HBB, HBG1, and HBG2 mRNA levels in proliferating clonal cell lines. HBB, HBG1, and HBG2 mRNA expression was normalized to actin and displayed as a percentage of HBB + HBG1 + HBG2. (D) Same as panel C but the cells were differentiated for 10 days. (E) Schematic representation of the primers used in the RT-qPCR analysis of the HBG transcripts (top). The fold-change, normalized for primer efficiency based on DNA amplification with HBG2 exon 2 set to 1, shown in arbitrary units for a ΔBCL11A clone and 2 Del2Rec deletion clones (bottom). (F) HbF of 2 clonal Del2Rec deletion cell lines, as measured by HPLC. The percentage of HbF calculated over the total Hb tetramers. (G) Representative flow cytometry plots showing the percentage of HbF positive cells in proliferating clonal cell lines (top row) and after 10 days of differentiation (bottom row). (H) The ATAC-seq tracks of the HBB gene cluster in differentiated (10 days) WT HUDEP-2 cells and 2 Del2Rec clones. The horizontal dashed lines indicate the deletion generated in Del2Rec clones. The signal was scaled to the genome-wide TSS signal. Max scales were set to the highest scaled signal in the HBB locus. (I) H3K4me3 CUT&RUN tracks of the HBB gene cluster in 2 differentiated WT HUDEP-2 samples and 2 Del2Rec clones. The horizontal dashed lines indicate the deletion generated in Del2Rec clones. Scaling is based on the average coverage at the HBA locus. WT, wild-type.

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