X chromosome inactivation (XCI) assay
The HUMARA (Human Androgen Receptor) assay (Allen, Zoghbi, Moseley, Rosenblatt, & Belmont, 1992; Bertelsen, Tumer, & Ravn, 2011; Echevarria et al., 2016) was used to determine the ratio of inactivated paternal vs maternal X chromosomes. Briefly, gDNA samples (0.25 ug) were incubated with 10 U HpaII (New England Biolabs) at 37°C overnight followed by heat inactivation of the enzyme at 80°C for 20 min. The highly polymorphic androgen receptor promotor region of the HpaII treated samples and untreated control samples were amplified with 0.2 μM forward primer 5’-FAM -TCCAGAATCTGTTCCAGAGCGTGC-3’ and 0.2 μM reverse primer 5’-TCCAGAATCTGTTCCAGAGCGTGC-3’ with 0.2 mM dNTP, 1.5 mM MgCl2 and 0.025 U AmpliTaq Gold DNA Polymerase (Thermo Fisher) by PCR amplification (initial denaturation at 95°C for 6 min followed by 35 cycles of 40 s at 95°C, 30 s at 60°C, and 1 min at 72°C and a final extension step at 72°C for 2 min). After adding the GeneScan 500 LIZ dye Size Standard (Thermo Fisher), the mixtures were analyzed on an ABI 3730 DNA Analyzer with GeneMapper software 3.7 (Applied Biosystems). XCI ratio was estimated by the area of the smaller base size (XC1) and larger base size (XC2) peaks, with formula as follows: peak area of [(XC1digested/XC1nondigested) / ((XC1digested/XC1nondigested) + (XC2digested/XC2nondigested )) ].