2 primary cell 4d nucleofector x kit

Mouse primary dermal fibroblasts were established from neonatal C57BL/6 Tmc1^WT/WT and Tmc1^Bth/WT animals. Briefly, after euthanasia, a small amount of skin was dissected into small pieces and washed with PBS. Next, cells were treated at 37 °C with 1 mg/ml collagenase I (Worthington) for 30 minutes followed by 0.05%-trypsin-EDTA treatment for 15 minutes. Cells were cultured in 10% fetal bovine serum containing DMEM (Gibco) supplemented with 1x penicillin/streptomycin (Gibco). Cell lines were validated by Sanger sequencing (see below). Mycoplasma screening (MycoAlert, Lonza, Basel) was performed regularly and before transfection experiments. For transfection of fibroblasts, we used Nucleofection (Lonza) (CZ-167 program, P2 Primary Cell 4D-Nucleofector X Kit). Every transfection reaction was performed in duplicate and experiments were performed on at least two separate occasions. Four days after transfection of pX458 plasmids, cells were sorted based on GFP fluorescence using a FACS Aria Cell Sorter (BD), and genomic DNA was isolated and analyzed by Sanger sequencing and targeted deep sequencing (see below). In the case of high fidelity SpCas9s (eSpCas9(1.1), HypaCas9, SpCas9-HF1) and SaCas9-KKH transfections, cells were not sorted, but genomic DNA was isolated from all cells 4 days after transfection for deep-sequencing analysis.