Androgen Receptor and MECP2 Allele Analysis

Androgen receptor (AR) assay was performed as previously described [10 (link)]. In brief, 400 ng of genomic DNA from iPSCs/ESCs was digested by CpG methylation-sensitive restriction enzymes HpaII and HhaI for 6 h. 2 μL of digestion was amplified using Platinum Taq DNA Polymerase High Fidelity (Invitrogen) with AR gene primers with a FAM label (Table S2). Male samples were used as a control to confirm complete digestion. Electrophoresis was performed by TCAG at The Hospital for Sick Children. Traces were analyzed using PeakScanner (Thermo Fisher Scientific). To identify which MECP2 allele was expressed on the active X chromosome, RNA was isolated from iPSCs and reverse transcribed into cDNA using SuperScript III (Invitrogen). Primers flanking the variant region (Table S2) were used for amplification with Platinum Taq DNA Polymerase High Fidelity or Q5 High Fidelity DNA Polymerase (New England BioLabs), followed by cloning as per the TOPO TA Cloning Kit (Invitrogen) or Zero Blunt TOPO Cloning Kit (Invitrogen) in OneShot TOP10 (Thermo Fisher Scientific) or Max Efficiency DH5α (Invitrogen) competent E. coli strains. 5–10 bacterial colonies per iPSC cell line tested were picked and grown in LB Medium (MP Biomedicals) for DNA extraction with Quick Plasmid MiniPrep Kit (Invitrogen). Samples were Sanger sequenced at TCAG and aligned to WT MECP2 template using benchling.com.