DNA was isolated from pluripotent stem cells and neurons at indicated times by the salting out method [
39], and the CGG-repeat number in SC120 iPSCs and WCMC37 hESCs and lineages derived from these cells were analyzed by RPT-PCR and MS_RPT-PCR respectively as described before [
40]. Briefly, 300 ng of genomic DNA was digested with either Fast-HindIII (Thermo Fisher Scientific) or HindIII with HpaII (33 units/μg DNA) (New England Biolabs, Ipswich, MA) overnight in 22.5 μl volume containing 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl
2, 22 mM (NH
4)
2SO
4, and 0.2 % Triton X-100. Total 5 μl of digested DNA from both samples were processed for the repeat PCR with 15-μl PCR reaction mix containing a final composition of 50 mM Tris-HCl (pH 8.9), 1.5 mM MgCl
2, 22 mM (NH
4)
2SO
4, 0.2 % Triton X-100, 0.5 μM of the primers Not_FraxC (5′-AGTTCAGCGGCCGCGCTCAGCTCCGTTTCGGTTTCACTTCCGGT-3′) and Not_FraxR4 (5′-CAAGTCGCGGCCGCCTTGTAGAAAGCGCCATTGGAGCCCCGCA-3′), 2.5 M betaine (Sigma-Aldrich), 2 % DMSO (Sigma-Aldrich) and 0.27 mM each dNTP (New England Biolabs), and 0.4 units of Phusion® DNA polymerase (New England Biolabs). The samples were then loaded onto a preheated (>70 °C) PCR block (C1000-Touch, Bio-Rad, Hercules, CA) and subjected to the following cycles of heating and cooling: 98 °C for 3 min, 30× (98 °C for 30 s, 64 °C for 30 s, 72 °C for 210 s) and 72 °C for 10 min. The PCR products were resolved on TAE-1 % agarose gels and visualized with ethidium bromide according to standard procedures. For samples analyzed on capillary electrophoresis, Not FraxR4 primer was FAM labeled. For the HT14 iPSCs, the CGG-repeat size was determined as previously described [
41] except that 4, 7, 2′, 4′, 5′, 7′-hexachloro-6-carboxyfluorescein (HEX)-labeled primer frax-F (5′-AGCCCCGCACTTCCACCACCAGTCTCCTCCA-3′) and primer frax-C (5′-GCTCAGCTCCGTTTCGGTTTCACTTCCGGT-3′) were used. This primer pair produces a PCR product that contains the repeat and 121 bp of DNA from the upstream flank and 100 bp from the downstream flank for a total of 221 bp of the flanking DNA. The
FMR1 promoter methylation was assessed by quantitative methylation-specific PCR (qMS-PCR) essentially as described before [
40]. Briefly, 300 ng of DNA was sonicated into <500-bp fragments using a Bioruptor® (Diagenode, Denville, NJ) and digested overnight with HpaII or mock digested. Quantitative PCR using 2 μl of the mock-digested or HpaII-digested DNA was then carried out in 5 replicates using Power SYBR® green master mix on StepOnePlus™ Real-Time PCR machine (Thermo Fisher Scientific) in a final volume of 20 μl. The percentage of DNA methylation was determined by averaging the results of five technical replicates and calculating the difference in the Ct values of the digested and mock-digested samples using the ∆∆Ct method.
FMR1 promoter region was amplified using primers
FMR1 ex 1 (F) (
5′-GAACAGCGTTGATCACGTGAC-3′) and
FMR1 ex 1 (R) (
5′-GTGAAACCGAAACGGAGCTGA-3′). The efficiency of HpaII digestion was assessed by amplification of an unmethylated region of
GAPDH using primers
GAPDH exon1 (F) (5′-TCGACAGTCAGCCGCATCT-3′) and
GAPDH intron1 (R) (5′-CTAGCCTCCCGGGTTTCTCT-3′). Additionally, DNA methylation at 22 CpG residues in the
FMR1 promoter in WCMC37 p45 cells was also analyzed by pyrosequencing of bisulfite-converted DNA by EpigenDx, Inc. (Hopkinton, MA) using assays ADS1451-FS1 and ADS1451-FS2.