Fig. 2

CGG-repeat instability in FX ESCs. a CGG-repeat size and methylation analysis for the WCMC37 ESCs and the individual lineages derived from it was done by MS_RPT-PCR followed by agarose gel electrophoresis as described in the “Methods” section. The “+” and “−“ signs indicate the presence or absence of predigestion by the methylation-sensitive restriction enzyme, HpaII. M, 100-bp DNA size ladder and rpts, CGG repeats. b Pyrosequencing analysis of DNA methylation in the FMR1 promoter of WCMC37 and 37A cells at passage 44 and 48, respectively. c qMS-PCR analysis of DNA methylation in the FMR1 promoter of the samples analyzed in a. The extent of methylation was determined by the ∆∆Ct method and the individual technical replicates varied by <0.3 Ct. d The abundance of total histone H3, H3K9me2, H3K9me3, H3K27me3, and H4K20me3 in the FMR1 exon1 region is shown relative to GAPDH. Data shown are an average of two independent experiments and error bars represent standard deviation. The 37A line at passage 59 (p59) showed the presence of mostly active FMR1 alleles with only 15 % DNA methylation, consistent with the low levels of repressive histone marks on the FMR1 exon1 compared to WCMC37 cells that carry a silenced FMR1 allele. The passage number of the cells used in panels a and c are as follows: WCMC37 p44, 37A p48, 37B p53, 37C p55, 37D p55, 37 F p55, 37E p55. See Additional file 1: Figure S1a for pluripotency marker staining in WCMC37 cells