i3N iPSC culture and neuronal differentiation

We modified a well-characterized control iPSC line (WTC11) that harbors a dox-inducible NGN2 transgene at the AAVS1 locus (i³N) (62, 63). dCas9-BFP-KRAB was stably expressed in these i³N iPSCs via TALEN-mediated integration of a CAG-dCas9-BFP-KRAB expression cassette into the CLYBL safe harbor locus (55). The dCas9-BFP-KRAB iPSCs were transduced with lentivirus expressing HP1α-sgRNA for 3 days and then selected by the addition of puromycin. To differentiate i³N dCas9-BFP-KRAB iPSCs expressing HP1α sgRNA into neurons, iPSCs were dissociated by using Accutase (#AT-104, Innovative Cell Technologies) and then seeded onto dishes coated with Matrigel (354230, Corning). Three days after differentiation, cells were dissociated by using Accutase and then seeded onto poly-l-ornithine–coated plates (6-well plate) or glass coverslips (24-well plate) at a density of 7 × 10⁵ or 2 × 10⁴ cells per well, respectively. Six days later, the neurons were fixed with 4% paraformaldehyde for immunofluorescence staining or harvested for Western blot and qPCR analyses.