FECH gene marker site (Single-plex assay design)

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FECH gene marker site (Single-plex assay design) Same forward primer with the degenerate base denoted by Y Assay for methylated sequences Y CG CG CG CG VIC MGB Probe targeting 2 of the 4 CpG sites within the DMR Different reverse primer targeting 2 of the 4 CpG sites near the 3’ end of the DMR Same forward primer with the degenerate base denoted by Y Assay for unmethylated sequences Y TG TG TG TG FAM MGB Probe targeting 2 of the 4 CpG sites within the DMR Different reverse primer targeting 2 of the 4 CpG sites near the 3’ end of the DMR B Ery-1 & Ery-2 marker sites (Duplex assay design) Same forward primer with the degenerate base denoted by Y Y CG CG CG VIC MGB Probe targeting the 3 CpG sites within the DMR Same reverse primer Assay for both methylated and unmethylated sequences Same forward primer with the degenerate base denoted by Y Y TG TG TG FAM MGB Same reverse primer Probe targeting the 3 CpG sites within the DMR Supplemental Fig. 6. Schematic diagram of the design of digital PCR assays targeting the three erythroblast-specific DMRs: (A) FECH-gene marker site (B) Ery-1 and Ery-2 marker sites.