Neuronal culture and treatment:

Procedures for rat neuronal culture were reviewed and approved for use by the Broad Institutional Animal Care and Use Committee, in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals. In each of N=4 biological repeats, 1–2 Embryonic Day 18 embryos were collected from a separate pregnant Sprague Dawley rat killed by CO₂ (Taconic). Embryo hippocampi were dissected in 4°C Hibernate E supplemented with 2% B27 supplements and 100 U/ml penicillin/strep (Thermo Fisher Scientific). Hippocampal tissues were digested in Hibernate E containing 20 U/ml papain, 1 mm L-cysteine, 0.5 mm EDTA (Worthington Biochem), and 0.01% DNase (Sigma-Aldrich) for 8 min. Neurons were centrifugated at 1000rpm by 5min, pellet with cells were then resuspended into NbActiv1 (BrainBits LLC, now TransnetYX) supplemented with 25mM glutamate, and plated at a density of 15,000 cells/well onto poly-d-lysine-coated, black-walled, thin-bottomed 96-well plates (Corning BioCoat). After 48 hours, AraC was added to each culture at a concentration of 1uM, to suppress glia proliferation and minimize well-to-well variability resulting from it. At DIV 5, the media was entirely replaced with warm NbActiv4. At DIV 6, each culture was treated with Accell SMARTpool (Dharmacon/Horizon from Perkin Elmer), a mix of four chemically modified self-transfecting siRNAs, against the relevant gene (Table S3) to a total siRNA concentration of 1uM in NbActiv4. Cultures were then left undisturbed until fixation on DIV 21. Each plate included 60 wells/separate cultures, 3–4 in each treatment group. Across 4 plates, one for each biological repeat, this results in a total of n=11–18 technical repeats in each treatment group. For validation experiments, cultures were treated at DIV 6 with 0.1uM/0.5uM bpV(pic), 0.2uM/2uM Harmine, 20uM/50uM D-AP5, and Nontargeting, Shank3 or Grin2a Accell SMARTpool siRNA, and left undisturbed until fixation on DIV 8 or 19 as described.