NPCs per well (3.0 × 106) were seeded onto growth factor-reduced Matrigel-coated six-well plates in NPC medium. The following day, lentiviruses generated as above using either the lentiviral vectors dCas9:VP64-T2A-Puro, dCas9:VPR-T2A-Puro, and dCas9:KRAB-T2A-Puro were added, and cultures were spinfected (1 hr, 1,000 × g, 25°C). Following spinfection, plates were transferred to a cell culture incubator for 3 hr. Medium was then removed and replaced with fresh NPC medium. Two days later, fresh NPC medium containing 1 μg/mL puromycin (Sigma, no. P7255) was added and cells were expanded in NPC medium containing 1 μg/mL puromycin followed by banking in liquid nitrogen. Once thawed, NPCs were grown in NPC medium containing 1 μg/mL puromycin for the remainder of the experiment. Antibiotic-selected NPC lines were validated via FACS using an antibody for Cas9. Vendors, catalog numbers, and dilutions of all antibodies used are listed in Table S4.
