Allele-specific mutation bias is caused by allele-specific chromatin modifications.

(A) The degree of methylated alleles correlates with allele-specific mutation bias across three imprinted target sites (sgInpp5f_v2, sgKvDMR1, sgImpact) in two mESC lines. Each point represents the mean enrichment of mutations on paternal alleles over biological triplicate editing experiments (y-axis) and a single CpG methylation measurement from mock-transfected cells (x-axis). (B) Schematic illustrating the derivation of high-passage cells in order to induce LOI. Media composition (1i, 2i) and cell culture protocols are detailed in Materials and methods. (C) CpG methylation levels at three imprinted loci in low- and high-passage mESCs (B×J only) following mock transfection. Note that only the Inppf5_v2 locus has undergone extensive demethylation during culture. (D) Allele-specific mutation frequencies in low- versus high-passage cells. Error bars represent SD (n = 3) Transfectants were not selected in these experiments, which accounts for the lower overall mutation frequency relative to Fig 2. Transfection was also less efficient in high-passage cells. (E) Stacked histograms show the allelic mutation bias in each experiment. Asterisks denote p-values for unpaired t tests on the fold-difference between mutation frequencies on maternal and paternal alleles in low- compared to high-passage cells. ***p S1 Data, and details of MiSeq libraries including SRA accessions are provided in S2 Data. LOI, loss of imprinting; mESC, mouse embryonic stem cell; SRA, Sequence Read Archive.