Supplementary Figure 1

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1 Supplementary Figure 1 5 microns C7 B6 unclassified H19 C7 signal H19 guide signal H19 B6 signal C7 SNP spots H19 RNA spots B6 SNP spots colocalization H19 RNA classification Supplementary Figure 1. Allele-specific in wild-type MEFs. Micrographs demonstrating allele-specific detection of B6 and C7 alleles in a representative B6xC7 MEF with a genotype opposite from that found in Figure 1B. Demonstrated from left to right in the three large micrographs as well as in a representative region below are H19 C7 maternal probe, guide probe, and B6 paternal probe. The representative region shows how colocalization enables RNA classification. Under each micrograph are the computationally detected spots corresponding to single RNA transcripts. We labeled the guide probes with Cal Fluor 61, and C7 and B6-specific SNP FISH probes targeting the 5 SNPs between the two alleles with Cy5, Cy3, respectively. All scale bars correspond to 5 µm.

2 Supplementary Figure 2 A 5.5% 4.7% B6xB6 4.6% 4.2% 47.% 4.2% 4.% B6xC7 44.9% C7 B6 undetected 3-color 4.1% 2.5% 4.3% 2.2% 43.8% 45.7% 49.6% 47.8% C7xC7 B False colocalization after applying shift 4.1% 2.5%.6% 2.5% 43.8% 1.8% 49.6% 86.1% C 11.8% 2.9% 15.% 22.9% 37.9% 29.4% H19 +/-DMD9CG mutant 32.5% Dye Swap 29.5% Supplementary Figure 2. Allele-specific detection is not affected by fluorophore and pixel colocalization. A. Efficiency of H19 mrna identification (B6, C7, neither or both) for wild-type MEFs of 4 different genotypes. B. Pixel shift demonstrates loss of colocalization of probes in wild-type MEFs. C. Overall of H19 in MEFs from the H19 +/DMD-9CG mutant mouse compared with overall with swapped fluorophores, which shows comparable ratios independent of dye effects.

3 Supplementary Figure 3 A 3 C7 x B6 WT MEF B 4 C7 x B6 9CG MEF Total H19 RNA Count 2 Total H19 RNA Count C 4 D 4 Total H19 RNA Count 3 2 p =.34 Total H19 Transcript Counts R = Detected H19 Transcript Counts Supplementary Figure 3. Determination of monoallelic and biallelic ratios in wild-type and mutant MEFs. A. Scatter plot of allele-specific of wild-type MEFs. Almost all cells have a H19 C7/total H19 ratio greater than.8, suggesting.8 as an adequate cutoff for monoallelic H19. B. The H19 C7 distribution and total H19 RNA for H19 +/DMD-9CG mutant MEFs. We classified cells with an H19 C7/ total H19 ratio greater.8 as monoallelic. C. Box plot showing H19 RNA count distributions between monoallelic and biallelic cells in H19 +/DMD-9CG mutant MEFs. cells express approximately 5% more RNA on average than monoallelic cells (biallelic n = 12, monoallelic n = 31, p =.34 by bootstrapping through cell label permutation). D. Scatterplot showing linear relationship between detected H19 transcript counts and total H19 transcript counts in H19 +/DMD-9CG mutant MEFs.

4 Supplementary Figure 4 A mrna counts B 6 4 mrna counts H19 C7 H19 B6 Igf2 H19 C7 H19 B6 Igf2 C 3 C x B WT MEF IGF2 D C x B 9CG MEF IGF2 6 Igf2 mrna Count 2 Igf2 mrna Count Supplementary Figure 4. Igf2 in monoallelic and biallelic MEFs. A. Igf2 (green dots) and H19 (horizontal bars) in wild-type MEFs. B. Igf2 (green dots) and H19 (horizontal bars) in H19 +/DMD-9CG mutant MEFs. C. Scatterplot showing H19 C7 bias versus total Igf2. Most cells have a C7/total H19 ratio greater than.8, suggesting.8 as the cutoff for monoallelic H19. D. The H19 C7 distribution and total Igf2 RNA for H19 +/DMD-9CG mutant MEFs. Only monoallelic cells express Igf2 but not all monoallelic cells express Igf2.

5 Supplementary Figure single cells 2 C7 B6 Supplementary Figure 5. MEF clones demonstrate that monoalleic and biallelic H19 is heritable across cell divisions. H19 +/DMD-9CG mutant MEF colonies from a second independent experiment (larger replicate of experiment in Fig. 3B).

6 Supplementary Figure 6 5 μm H19 C7 SNP probe H19 guide probe H19 B6 SNP probe Igf2 probe Supplementary Figure 6. Allele-specific in cardiac tissue from H19 +/DMD-9CG mice with Igf2 co. Regions of monoallelic H19 (magenta) and biallelic H19 (yellow). Igf2 (green) expresses only in regions of monoallelic H19.