Feline Lindhout

Quantitative mapping of transcriptome and proteome dynamics during polarization of human iPSC-derived neurons 2 53 the sCMOS chip with no intermediate lens at a magnification of 0.063 µm/pixel at 100x. All imaging was performed in full conditioned differentiation (day 5) or maintenance (day 7 or 13) medium for hiPSC-derived neuron cultures (Axol Bioscience), and cells were kept at 37°C with 5% CO2 using a stage top incubator (model INUBG2E-ZILCS, Tokai Hit). For analysis, kymographs were generated using the FIJI plugin KymoResliceWide v.0.4 (https://github.com/ekatrukha/KymoResliceWide ), and parameters of microtubule plus-end dynamics were determined by manually tracing microtubule growth events. Electrophysiology A 12 mm coverslip containing hiPSC-derived neurons (7–14 days after plating) was transferred to the microscope recording chamber before the start of each experiment. Coverslips were continuously perfused with carbonated (95% O2, 5% CO2) artificial cerebrospinal fluid (ACSF, in mM: 126 NaCl, 3 KCl, 2.5 CaCl2, 1.3 MgCl2, 26 NaHCO3, 1.25 NaH2PO4, 20 glucose; with an osmolarity of ~310 mOsm/L) at a rate of approximately 1 ml/min. As an acute change in extracellular osmolarity has previously been reported to affect the exciTable properties of neurons, an extra medium refreshment was done the day before recording (Pasantes-Morales 1996). Bath temperature was monitored and maintained at 30-32 °C throughout the experiment. Recording pipettes (resistance of 4-7 MΩ) were pulled from thick-walled borosilicate glass capillaries (World Precision Instruments) and filled with internal solution (in mM: 140 K-gluconate, 4 KCl, 0.5 EGTA, 10 HEPES, 4 MgATP, 0.4 NaGTP, 4 Na2-Phosphocreatine; with pH 7.3 and osmolarity 295 mOsm/L), containing 30 µM Alexa 568 (Thermo Fisher Scientific) to facilitate visualization of cells. For post hoc cellular identification, biocytin was included in the internal solution. On an upright microscope, hiPSC-derived neurons were visually identified with a 60x water immersion objective (Nikon NIR Apochromat; NA 1.0) and selected for whole-cell somatic patch clamp recordings. Cells were kept at a baseline holding potential of -60 mV in both voltage and current clamp throughout the recording. Data was analyzed with pCLAMP software and custom-written MATLAB scripts. Sample preparation RNA sequencing ~100,000 hiPSC-derived NPCs were plated per well for bulk RNA sequencing samples. Prior to sample preparation, all equipment and surfaces were cleaned with RNaseZap (Sigma-Aldrich). Replicates of hiPSC-derived neurons were harvested at three different timepoints of differentiation (days 1, 3, and 7) with 200 µl Trizol (Invitrogen) per sample and stored at -80 °C until sequencing. RNA extraction, cDNA library preparation (CEL- Seq2 protocol), quality control for aRNA and cDNA, and sequencing on a NextSeq500 High output 1x75 bp paired end run with 2% sequencing depth were performed by Single Cell Discoveries (Utrecht, The Netherlands). Bioinformatic analysis RNA sequencing Mapping to reference transcriptome Hg19 was performed by Single Cell Discoveries