Hypoxia increases overall 5-hmC density during in vitro erythroid differentiation. (A) Mass spectrometry quantification of 5-mC relative to all cytosine species during erythroid differentiation. (B) Mass spectrometry quantification of 5-hmC relative to all cytosine species during erythroid differentiation. Quantification of 5-hmC peaks by peak counts (C) and by total bases covered by peaks (D) in normoxia vs hypoxia at days 3, 7, and 10. Normalized to corresponding measurements at day 0. Day 0 peak count was 473 206 for replicate 1 and 185 665 for replicate 2. (E) Dot plots of numbers of sequencing reads within peaks called by MACS2 per million total aligned reads. Each dot represents a genomic region of 1 Mb. (F) Similar to panel E, global measurement of sequencing reads within peaks, normalized to day 0. (G) Box plot of all 5-hmC peaks FPKM in all time points in normoxia vs hypoxia. Wilcoxon rank-sum test was used to evaluate statistical significance. ^‡P < 10⁻⁸. Quantification of peaks that gained or lost 5-hmC in hypoxia by peak counts (H) and by total bases covered by peaks (I) as percentages of total peaks. N = 2 for mass spectrometry and 5-hmC pull-downs. *P < .05.