sequencing of GuideSeq from HEK293T cells: HyperDrive_VEGFA_site_3
HEK293T cells were cultured at 37C under humidified 95% air/5% CO2 in Dulbecco's modified Eagle's medium (DMEM) containing glucose (4.5 g/l), 1 mM sodium pyruvate, 2 mM glutamine and 10% fetal bovine serum (FBS). Cells were seeded at 60% confluence in 6-well plates and transfected after 24 h with 2 g of plasmid DNA and 500 ng of annealed repair oligos in OptiMEM media (Gibco, Life Technologies) using a 1:1 mix of two transfection reagents (Lipofectamine LTX Reagent with Plus Reagent, Invitrogen, and FuGene HD, Promega). Genomic DNA was harvested after 72 h using a Zymo Quick-DNA Miniprep Plus Kit and GUIDE-seq library preparation was performed as described by Malinin, N.L., Lee, G.H., Lazzarotto, C.R., Li, Y., Zheng, Z., Nguyen, N.T., Liebers, M., Topkar, V. V., Iafrate, A.J., Le, L.P., et al. (2021). Defining genome-wide CRISPRCas genome-editing nuclease activity with GUIDE-seq. Nature Protocols 2021 16:12 16, 55925615. https://doi.org/10.1038/s41596-021-00626-x., with minor modifictions as follows. DNA fragmentation and end-repair were performed using an NEBNext Ultra II FS DNA Module (New England Biolabs) and purified fragments were ligated to Y-adapter oligonucleotides using an NEBNext Ultra II Ligation Module.