Genotyping of single cell expanded genome edited hPSCs clones by RLFP

The RFLP analysis was performed as previously described Hernandez et al., 2005 following standard protocols (Green and Sambrook, 2012) to screen single-cell expanded clones for the insertion of the LRRK2 (G2019S) mutation. Genomic DNA was amplified with primers SP-LRRK2-RLFP and ASP-LRRK2-RLFP (Supplementary file 1) under standard PCR conditions followed by restriction digest with SfcI. The LRRK2 (G6055A, G2019S) mutation at coding nucleotide 6055 of LRRK2 creates a novel SfcI cleavage site which allows to distinguish the two alleles after separation on a 3% agarose gel with the reference allele generating fragments of 228 bp and 109 bp and the mutated allele generating fragments of 207 bp, 109 bp, and 21 bp. Mutation carrying clones were further analyzed using Sanger and NGS sequencing of the PCR product.