amplicon sequencing of HEK293T cells: TurboABE_HEK293T_FANCF_site_2_rep3
HEK293T cells were obtained from ATCC (CRL-3216) and cultured at 37C under humidified 95% air/5% CO2 in Dulbeccos modified Eagles medium (DMEM) containing glucose (4.5g/l), 1mM sodium pyruvate, 2mM glutamine and 10% fetal bovine serum (FBS). Cells were seeded at 60% confluence in 24-well plates and transfected after 24h with 500ng of DNA 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). Cells were trypsinized 72h post transfection the and the cell pellets lysed for DNA extraction using the KAPA Express Extract Kit, according to the manufacturers instructions (Sigma-Aldrich). Amplicons were generated using primers flanking the gRNA target site and incorporating Illumina adaptor sequences.