Nucleofector 2b device kit 5

A375 cells were obtained from American Type Culture Collection (cat #: CRL-1619, ATCC, Manassas, VA) and maintained for up to 15 passages in DMEM supplemented with 10% FBS (cat #: A3160401, Life Technologies, Carlsbad, CA), GlutaMAX (Life Technologies), antibiotic–antimycotic (Life Technologies), and 20 mM HEPES pH 7.4. A Nucleofector 2b device (Kit V; Lonza, Basel, Switzerland) was used to transfect plasmids. Cells were plated on six-well glass-bottom plates (Cellvis, Mountain View, CA) either directly, after coating with 10 µg/mL human plasma fibronectin (cat #: FC010, Millipore, Billerica, MA), or with poly-L-Lysine (cat #: A-005-C, Millipore), as noted in each figure.

Zinc finger nuclease activity was assessed by measuring disruption of an integrated, constitutively expressed eGFP reporter in a clonal U2OS cell line previously described^{41 (link)}. Cells were cultured in DMEM supplemented with 10% FBS, 2 mM GlutaMax (Life Technologies), 1% penicillin/streptomycin, 1% MEM nonessential amino acids (Life Technologies), 2 mM sodium pyruvate and 400 μg ml^–1 G418. One microgram of each ZFN monomer plasmid DNA and 200 ng of ptdTomato-N1 plasmid DNA were transfected in duplicate into 5 × 10⁵ cells using a Lonza Nucleofector 2b Device (Kit V, Program X-001). In each assay 2 μg of parental empty vector (a modified derivative of the JDS71 vector from Addgene) and 200 ng of ptdTomato-N1 were used as a negative control, and 2 μg of a dual-spCas9-guide-expressing vector (modified Addgene plasmid no. 41815) and 200 ng of ptdTomato-N1 as a positive control, in each experiment. Cells were grown in six-well dishes for 3 days post transfection, harvested, maintained on ice and analyzed for expression of eGFP and tdTomato on a Sony SH800 cell sorter. To restrict the analysis to cells that probably received both ZFN monomer plasmids, populations were first gated on the top 15–25% tdTomato⁺ cells then analyzed for loss of eGFP expression (Supplementary Fig. 11).