Extended Data Fig. 1: Validation of custom allele-specific RT–qPCR assays and dose-dependent ZFP protein expression.

a, Validation of the rs363099 RT–qPCR assay. GM02151 (CAG18/45) and GM04723 (CAG15/67) fibroblasts are heterozygous for the SNP rs363099T/C. In these lines, the rs363099T variant is in phase with normal HTT and rs363099C is in phase with mHTT. Separate plasmid templates harboring the rs363099C or rs363099T amplicons were generated. The rs363099C template was serially diluted twofold, from 5 to 0.156 femtograms (fg), in the presence of a fixed amount (1.25 fg) of the rs363099T template (ratio of rs363099C/rs363099T, 0.125/1–4/1; left panel). Conversely, the rs363099T template was serially diluted twofold, from 5 to 0.156 fg, in the presence of a fixed amount (1.25 fg) of the rs363099C template (ratio of rs363099T/rs363099C, 0.125/1–4/1; right panel). Plasmids were diluted into total cDNA prepared from fibroblasts for which HTT and mHTT expression had been repressed using a strong biallelic HTT repressor. Plasmid inputs were chosen so that the RT–qPCR signal from the 1/1 plasmid ratio gave approximately the same signal as HTT and mHTT in untreated cells. rs363099C- (red) or rs363099T-specific (blue) RT–qPCR assays (see Methods) were performed independently for each of the dilution samples. Relative quantities were scaled to the values obtained for the 1/1 ratio of the rs363099C and rs363099T templates. b, Validation of the rs362331 RT–qPCR assay. ND30259 (CAG21/38) fibroblasts are heterozygous for the C/T SNP rs362331. The rs362331T variant is in phase with normal HTT and rs362331C is in phase with mHTT. Separate plasmid templates harboring rs362331C or rs362331T amplicons were generated and evaluated in the same manner as in a using a reciprocal serial dilution range of 2–0.0625 fg in the presence of 0.5 fg of fixed template. rs362331C- (red) or rs362331T-specific (blue) RT–qPCR assays were performed independently for each of the dilution samples. Relative quantities were scaled to the values obtained for the 1/1 ratio of the rs362331C and rs362331T templates. a,b, n = 3 technical replicates; mean ± s.d. Similar results were replicated in at least one independent experiment for each assay combination. c, Representative cropped image of ZFP-A, -B and -C protein levels assessed by immunoblotting in GM04723 fibroblasts transfected at doses of 1,000, 300, 100, 30 and 10 ng ZFP-TF mRNA. Similar results were replicated in one independent experiment. The uncropped scans are provided as source data. d, Relative quantitation of protein levels from c. ZFP/GAPDH ratios for each sample were scaled to the signal from the 1,000-ng ZFP-C transfected sample.