sequencing of PCR mix for Homo sapiens:293T cell lines
PCR primers about 150bp upstream and downstream of on- and off-targets were designed with different barcodes and synthesized (Genescript). Then PCR amplification was performed using ~200 ng genomic DNA each with Takara LA Taq. Sequence specificity was verified by agarose gel electrophoresis and then purified using Universal DNA Purification Kit (Tiangen Biotech, Beijing, China). Amplicons with different barcodes were mixed together and DNA library was prepared using NEBNext Ultra II DNA Library Prep Kit (NEB). Deep sequencing was performed using Illumina Hiseq