Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 51 and every three days during further maintenance. Cells were kept in culture for maximum ~15 days to ensure high quality of the cultures. Lentivirus and lentiviral infection The constructs used for lentiviral transduction are FUGW-GFP (Addgene #14883, (Lois et al. 2002)) and Marcks-tagRFP-T-pIres-GCN4-MacF18. Marcks-tagRFP-T-pIRES-GCN4- MacF18 cloning is previously described (Yau et al. 2016). The construct was subcloned into the pSIN-TRE-mSEAP-hPGK-rtTA2sM2 lentiviral vector (kindly provided by Dr. Didier Trono, Ecole Polytechnique Fédérale de Lausanne, Switzerland) wherein the neuron- specific synapsin promotor has substituted the PGK promotor. Generation of lentiviral particles was performed as previously described (Yau et al. 2014). Lentiviral transduction of cells was performed two hours after plating. The tetracycline-dependent expression was induced by adding500 ng/ml doxycycline to the medium two days before imaging. Antibodies Primary antibodies used in this study: mouse-IgG1 anti-Nestin (1:200; MAB5326, Millipore), rabbit anti-Ki67 (1:500; ab92742, Abcam), mouse-IgG2b anti-β3-Tubulin (1:400; 082M4845, Sigma), chicken anti-MAP2 (1:2000; ab5392, Abcam), rabbit anti-Trim46 (1:500; homemade (van Beuningen et al. 2015)), mouse-IgG1 anti-AnkG (1:200; 33-8800, Life Technologies), mouse-IgG1 anti-PanNav (1:200; S8809, Sigma). Secondary antibodies used in this study: anti-rabbit Alexa 405 (A31556, Life Technologies), anti-mouse-IgG1 Alexa 488 (A21121, Life Technologies), anti-rabbit Alexa 568 (A11036, Life Technologies), anti-mouse Alexa 568 (A11031, Life Technologies), anti-mouse-IgG2b Alexa 594 (A21145, Life Technologies), anti-rabbit Alexa 647 (A21245, Life Technologies), anti-mouse Alexa 647 (A21236, Life Technologies), anti-mouse-IgG2a Alexa 647 (A21241, Life Technologies), anti-chicken Alexa 647 (A21449, Life Technologies). Immunofluorescence Cells were fixed for 5-10 min in PBS with 4% paraformaldehyde/4% sucrose at room temperature, and washed three times with PBS. For immunofluorescence stainings, fixed cells were sequentially incubated with primary and secondary antibodies dissolved in gelate dilution buffer (GDB; 0.2% BSA, 0.8 M NaCl, 0.5% Triton X-100, 30 mM phosphate buffer, pH 7.4), and washed three times with PBS after every antibody incubation step. Coverslips were mounted on glass slides using Vectashield mounting medium (Vector laboratories) with or without DAPI. Microscopy Fixed cells were imaged using a LSM700 confocal laser-scanning microscope (Zeiss) with a Plan-Apochromat 63x NA 1.4 oil DIC; EC Plan-Neofluar 40x, NA 1.3 Oil DIC; and a Plan-Apochromat 20x, NA 0.8 objective. Live-cell acquisition was performed using spinning-disk confocal microscopy on an inverted research microscope Nikon Eclipse Ti-E,