4d nucleofector with x unit attachment

For transfection-based editing experiments in HEK293Ts, cells were seeded on a 12-well plate at 80% confluence and co-transfected with 750ng of base editor (BE), 750ng of sgRNA expression plasmid, and 4.5μl of polyethyleminine (PEI; 1mg/ml). Cells were harvested for genomic DNA, 3 days post-transfection. For virus production, HEK293T cells were plated in a 6 well-plate and transfected 12 hours later (95% confluence) with a prepared mix in DMEM media (no supplements) containing 2.5μg of lentiviral backbone, 1.25μg of PAX2, 1.25μg of VSV-G, and 15μl of PEI (1mg/ml). 36hrs following transfection, media was replaced with target cell collection media and supernatants were harvested every 8-12hrs up to 72hrs post transfection. ESCs col1a1-targeting constructs were introduced via nucleofection in 16-well strips, using buffer P3 (Lonza Inc., V4XP-3032) in a 4D Nucleofector with X-unit attachment (Lonza Inc.). Two days following nucleofection, cells were treated with media containing 150ug/ml Hygromycin B and individual surviving clones were picked after 9-10 days of selection. Two days after clones were picked Hygromycin was removed from the media and cells were cultured in M15 thereafter. To confirm integration at the col1a1 locus we used a multiplex col1a1 PCR ²⁸ .