Rnasep reference assay

Digital PCR reactions were prepared as follows: 1.5 μL cell lysate, 8.7 μL QuantStudio 3D Digital PCR Master Mix V2 (Thermo Fisher Scientific), 0.87 μL RNaseP reference assay (Thermo Fisher Scientific), 0.87 μL mNG2_1-10 or AmpR assay (prepared as described above), and 5.46 μL water. The amount of lysate was calculated based on the number of cells used and the dynamic range of the QuantStudio 3D system (400–4000 copies/μL), and input lysate volumes were adjusted as needed to obtain data within this dynamic range. 14.5 μL of the reaction mix was loaded onto dPCR chips using the QuantStudio 3D Digital PCR 20 K Chip Kit V2 (Thermo Fisher Scientific), taken through PCR thermocycling and analyzed on the QuantStudio 3D system (Thermo Fisher Scientific) according to manufacturer’s instructions. Data was analyzed using the QuantStudio 3D AnalysisSuite Cloud Software (Thermo Fisher Scientific).

Genomic DNA was extracted from monoclonal cells using DNeasy Blood and Tissue kit (Qiagen) following the manufacturer's instructions. Subsequently, qPCR-based copy number assay was carried out using Taqman Copy Number Assay and RNAse P Reference Assay (Thermo Fisher Scientific). qPCR probes specific to the GFP reporter gene was custom ordered from Thermo Fisher Scientific. Reactions were run using Quantstudio 7 Real-Time PCR system (Applied Biosystems), and subsequently analyzed for copy number using CopyCaller software (Applied Biosystems). For the inverse PCR, extracted genomic DNA was digested using a range of restriction enzymes including EcoRI, BamHI, SphI and NcoI (NEB) and ligated using T4 DNA ligase (NEB). Then, ligated circular DNAs were amplified using GFP reporter-specific primers (IDT) and Q5 hot start DNA polymerase (NEB). Amplified DNA was sequenced (Azenta) to identify the integration junction. Acquired sequences were compared with human genome sequence provided by UCSC Genome Browser (http://genome.uscs.edu)^{30 (link)}, using a genome assembly released in Dec 2013.