β mercaptoethanol

Skin biopsy samples were obtained from four individuals in a previously characterized American family, pedigree H¹² (Fig. 1a). C1 fibroblasts were from ATCC (CRL-2097). All studies followed institutional IRB, ISCRO and animal protocols approved by Johns Hopkins University School of Medicine. Informed consents were obtained from individuals from pedigree H. Mouse embryonic fibroblasts (MEFs) were derived from E13.5 CF-1 mouse embryos as previously described^{16 (link)}. Fibroblasts were cultured in Dulbecco’s modified Eagle’s medium (DMEM, Mediatech) supplemented with 10% fetal bovine serum (FBS, HyClone) and 2 mM L-glutamine (Invitrogen).
iPS cells were generated with the EBV-based vectors as previously described^{16 (link)}. Briefly, plasmids pEP4 EO2S ET2K (Addgene Plasmid 20927), pEP4 EO2S EN2L (Addgene Plasmid 20922), and pEP4 EO2S EM2K (Addgene Plasmid 20923) were transfected intohuman fibroblasts by Amaxa Nucleofector (Lonza; program U-023) at a concentration of 2 μg per 100 μl electroporation solution per 2 × 10⁶ cells. Colonies of iPS cells were manually picked after 3–6 weeks for further expansion and characterization. Lack of vector integration was confirmed by qRT–PCR analysis as previously described^{16 (link)}. Two lines from each individual that passed stringent criteria were used for the current study (Supplementary Table 1a). iPS cells (passage ≤ 35) were cultured on irradiated MEFs in human iPS cell medium consisting of D-MEM/F12 (Invitrogen), 20% Knockout Serum Replacement (KSR, Invitrogen), 2 mM L-glutamine (Invitrogen), 100 μM MEM NEAA (Invitrogen), 100 μM β-mercaptoethanol (Invitrogen), and 10 ng ml⁻¹ human basic FGF (bFGF, PeproTech) as described^{16 (link),31 (link)}. For feeder-free culture of iPS cells, cells were cultured on Matrigel (BD Biosciences) with mTeSR1 media (Stem Cell Technologies). Media were changed daily and iPS cell lines were passaged by collagenase (Invitrogen, 1 mg ml⁻¹ in D-MEM/F12 for 30 min at 37°C).
Karyotyping analysis by standard G-banding technique was carried out by the Cytogenetics Core Facility at the Johns Hopkins Hospital or Cell Line Genetics. Results were interpreted by clinical laboratory specialists of the Cytogenetics Core or Cell Line Genetics. Genotyping analysis was performed as described previously^{16 (link)}. Genomic DNA of fibroblasts and derived iPS cells was extracted by DNeasy Blood & Tissue Kit (Qiagen) following the manufacturer’s recommended protocol. Apair of specific primers was used to amplify the region around the 4-bp deletion (Supplementary Table 1c). PCR products were cloned by TA cloning and sequenced. Bisulphite genomic sequencing was carried out with the EZ DNA Methylation-Direct Kit (Zymo Research) as previously described^{32 (link)}. After bisulphite conversion of genomic DNA from iPS cells, primers specific to human OCT4 and NANOG promoters (Supplementary Table 1c) were used to amplify genomic DNA sequences with Platinum Taq DNA Polymerase High Fidelity (Invitrogen) for sequencing.
To assess the in vivo pluripotency of iPS cell lines, teratoma formation assays were performed^{16 (link)}. iPS cells were injected subcutaneously into the dorsal flank of SCID mice. Animals were monitored and teratomas were dissected at 8 to 10 weeks post-injection. Tissues were fixed in 10% neutralized formalin solution (Sigma). Embedding, sectioning and haematoxylin and eosin staining were carried out by the Pathology Core Facility at the Johns Hopkins University Hospital.
TALEN designs and constructions were based on a Golden Gate Assembly protocol with modifications to the vector backbone^{33 (link)}. Donor DNA vectors with a loxP-flanked PGK-hygromycin cassette were cloned between 5′ and 3′ homology arms (Fig. 3a), which were amplified from genomic DNA of a healthy subject and a patient with the DISC14-bp mutation. For targeting, TALENs (4 μg DNA of each plasmid) and linearized donor vectors (10 μg DNA) were electroporated into individual iPS cells (1 × 10⁶ to 2 × 10⁶ cells pretreated with 5 μM ROCK inhibitor, Y-27632, Cellagentech) using Nucleofector 2b (Lonza; program A-023). Transfected cells were transferred onto a 6-well dish pre-plated with inactivated MEFs and supplemented with Y-27632 in standard iPS cell medium. Positive colonies were selected by 10mg ml⁻¹ hygromycin B (Invitrogen) after 5 days of culture or until small colonies appeared. Resistant colonies were sub-cloned and expanded in 48-well plates. Over 200 clonal lines were screened. The loxP-flanked PGK-hygromycin cassette was removed by electroporation of a Cre recombinase expression vector (4 μg DNA). Specific integration, correct genetic editing and efficient removal of PGK-hygromycin cassette at each stage were verified by Sanger sequencing.