N2 supplement

NI media: Neural Induction medium consisting of DMEM/F12 (Thermo Fisher Scientific) with 1% N2 Supplement (Thermo Fisher Scientific), 1% MEM‐NEAA (Sigma Aldrich), 1% Glutamax (Thermo Fisher Scientific) and 1 μg/ml Heparin. Imp‐A: of 50% DMEM/F12 (Thermo Fisher Scientific), 50% Neurobasal (Thermo Fisher Scientific), 0.5% N2 Supplement (Thermo Fisher Scientific), 2% B27—Vitamin A (Thermo Fisher Scientific), 1% Glutamax (Thermo Fisher Scientific), 0.5% MEM‐NEAA (Sigma Aldrich), 50 μM 2‐ME solution, 1% Penicillin/Streptomycin (Sigma Aldrich) and 0.025% Insulin solution (Sigma Aldrich). Imp + A (HN): Imp‐A with 2.5 mM Ascorbic Acid, 2 g/l Bicarbonate (Sigma Aldrich). BP (LN): BrainPhys Neuronal Medium (Stem Cell Technologies), 2% B27 + A (50×, Thermo Fisher Scientific), 1% N2 Supplement (Thermo Fisher Scientific), 200 nM Ascorbic Acid (Sigma Aldrich), 0.2% CD Lipid Concentrate (Thermo Fisher Scientific), 7.4% glucose, and 1% Penicillin/Streptomycin.

The iPSCs were cultured according to a previously described protocol using our in-house Essential 8 medium^{25 (link)}. iPSCs from passages 25–35 were utilized for neural induction. The neural induction medium consisted of an equal ratio of DMEM F/12 and advanced neurobasal medium (Gibco, USA), 1X GlutaMAX (Gibco, USA), 1X B27 supplement without vitamin A (Gibco, USA), 1X N2 supplement (Gibco, USA), 10 µM SB431542 (Sigma-Aldrich, USA), 0.1 µM LDN193189, and 0.1X penicillin/streptomycin for differentiating iPSCs into neural progenitor cells (NPCs). Afterwards, neural expansion medium was used to culture the NPCs further (1:1 ratio of DMEM F/12 and Advanced neurobasal medium, 1X GlutaMAX, 1X B27 supplement without vitamin A, 1X N2 supplement, 20 ng/µl FGF-2, 20 ng/µl EGF, and 0.1X penicillin/streptomycin). The terminal differentiation was achieved on NPC passage 4–5 using neural differentiation medium, consisting of 1:1 ratio of DMEM F/12 and Advanced neurobasal medium, 1X GlutaMAX, 1X B27 supplement without vitamin A, 1X N2 supplement, 50 µM DB-cAMP, 200 µM Ascorbic acid, 20 ng/ml BDNF (Peprotech, USA), and 10 ng/ml GDNF (Peprotech, USA).

HeLa cells were maintained in DMEM media supplemented with 10 % fetal bovine serum. HCN-A94 adult rat hippocampal neural stem cells were obtained from Fred Gage (Salk Institute, La Jolla, CA, USA) and were maintained as described [25 (link)] in DMEM/F12 medium with N2 supplement and 20 ng/ml FGF-2 (Peprotech). Neuronal differentiation was induced in DMEM/F12 medium with N2 supplement (Life Technologies) and with 1 μM retinoic acid and 5 μM forskolin [26 (link)]. All cells were cultivated at 37 °C in a 5 % CO₂ atmosphere. Amlexanox (Tocris Bioscience) was dissolved in DMSO and applied to cells together with retinoic acid and forskolin.

The iCas9 hPSCs were gifted by Dr. Danwei Huangfu at Sloan-Kettering Institute and Dr. Jie Na at Tsinghua University. iCas9 hNSCs were differentiated from iCas9 hPSCs based on previous reports.^{49 (link)} Briefly, iCas9 hPSCs were cultured on matrigel-coated dishes and fed daily with mTeSR (STEMCELL) for 7 days. On the next day, mTeSR was substituted by N2 medium (DMEM/F12 supplemented with 0.5× N2 supplement (Gibco), 1 μM dorsomorphin (Tocris), and 1 μM SB431542 (STEMCELL)) for 1–2 days. hPSC colonies were lifted off, cultured in suspension on the shaker (95× rpm at 37 °C) for 8 days to form embryoid bodies (EBs) and fed with N2 media. EBs were then mechanically dissociated, plated on a matrigel-coated dish, and fed with hNSC maintenance medium (DMEM/F12 supplemented with 1× N2 supplement, 1× B27 supplement (Gibco), 1% penicillin/streptomycin, and 20 ng/mL bFGF (Gibco)). The emerging rosettes were picked manually, dissociated completely using Accutase (Gibco), and plated on a poly-ornithine/laminin-coated plate. The resultant hNSCs were expanded and maintained in the hNSC maintenance medium. The 293T cells were purchased from the cell resource center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences and cultured in DMEM medium with 10% FBS and 1% penicillin/streptomycin (Gibco).

The detection of prion seeding activity in samples was accomplished using the second-generation RT-QuIC assay [9 (link),10 (link)]. All samples were run in quadruplicate. The assay was performed in Nunc™ MicroWell™ 96-Well Optical-Bottom Plates (Thermo Fisher, Prague, Czech Republic) in 100 µL reaction mix for each well (10 mM phosphate buffer, pH 7.4; 300 mM NaCl; 10 µM thioflavin T (ThT); 1 mM EDTA; 0.002% SDS; and 0.1 mg/mL of rSHa PrP (90–231)). For the testing of brain homogenates (BHs), 2 µL of BH were added to 98 µL of reaction volume. BH samples were diluted 5 × 10⁻⁶ up to 5 × 10⁻⁹ in PBS buffer containing 1× N-2 supplement (Gibco, Thermo Fisher, Prague, Czech Republic N-2 supplement, 100×) and 0.1% SDS. CSF samples were tested undiluted or 10 times diluted in PBS (pH 7.4) and 15 µL of CSF sample were added into 85 µL reaction buffer. The reaction was carried out by FLUOstar Omega plate reader (BMG LABTECH GmbH, Ortenberg, Germany), undergoing repeating shaking cycles of 60 s (700 rpm, double orbital) and 60 s rest for 60 h at 55 °C. The fluorescence was measured every 15 min. Each test was analyzed by Mars software (BMG LABTECH GmbH, Ortenberg, Germany).

NSPCs were isolated from Arid1a WT and cKO forebrain at E16.5 as described previously.¹⁹, ²¹ Briefly, brain tissues were digested with TrypLE Express (Gibco, #12604013) in a 5% CO2 incubator at 37°C for 8 min. Then, 1 ml of DMEM/F‐12 containing 10% FBS, 1% GlutaMAX (Gibco, 35050061) and 1% Antibiotic‐Antimycotic (GIBCO, #15240‐062) was added into each sample to stop digestion. Single cells were obtained though scattering by repetitive pipetting and passing through 70 µm cell strainer. The single‐cell suspension was cultured with DMEM/F‐12 medium containing 1% N2 supplement (GIBCO, #17502‐048), 1% Antibiotic‐Antimycotic, 20 ng/ml basic fibroblast growth factor (FGF‐2, PeproTech), 20 ng/ml epidermal growth factor (EGF, PeproTech) in a 5% CO2 incubator at 37°C.
Differentiation of NSPCs assays was performed as previously.¹⁹, ²¹, ²² NSPCs were seeded on poly‐L‐ornithine/laminin‐coated coverslips at a density of 2 × 10⁵ cells/well. Following lentiviral infection for 48 h, NSPCs were incubated with DMEM/F‐12 medium containing 1% N2 supplement (GIBCO, #17502‐048), 1% Antibiotic‐Antimycotic, 5 µM forskolin (FSK, Sigma‐Aldrich, #F‐6886) and 1 µM retinoic acid (RA, Sigma‐Aldrich, #R‐2625) for 3 days. NSPCs were then fixed with 4% paraformaldehyde for 20 min and stained with Tuj1 antibody.