title: NextSeq 500 sequencing; RNA-seq of human neurons derived from H9 embryonic stem cell lines treated with BDNF, TrkB agonist ZEB 85 or NT4 against untreated controls
design: RNA-seq of human neurons derived from H9 embryonic stem cell lines treated with BDNF, TrkB agonist ZEB 85 or NT4 against untreated controls
library_construction_protocol: Human H9 ES cells were plated on matrigel-coated plates (Corning, VWR) and maintained in mTSER medium (STEMCELL Technologies) supplemented with 10 ng/ml of FGF2 (Peprotech). hES cells were passaged by manual dissociation with 0.02% EDTA in Dulbecco's PBS (0.5mM), pH 7.2 (Sigma) and seeded for an 8-day phase of neuronal induction in SLI medium which contained Advanced DMEM F-12 medium supplemented with glutamax, penicillin and streptomycin (ADF, Life Technologies) and 2% NeuroBrew-21 without retinoic acid (Miltenyi), the SMAD pathway inhibitors LDN193189 (1μM) and SB431542 (10μM), and the WNT pathway inhibitor IWR1 (1.5μM) (Tocris; [59]). Subsequently, neural progenitors were dissociated with Accutase (Life Technologies) and re-seeded for expansion to day 16 in LIA medium which contained ADF medium with 2% NeuroBrew-21 without retinoic acid (Miltenyi), LDN193189 (0.2μM), IWR1 (1.5μM) (Tocris) and 25 ng/ml of Activin A (Peprotech). Neuronal differentiation was initiated by seeding neural progenitors on a substrate of growth factor-reduced matrigel (Corning, VWR) & poly- L- lysine (Sigma) at a density of 100K cells/cm2 and cultured for 7 days in SynaptoJuice A medium which contained Advanced DMEM:F12 (with Glutamax), 1% penicillin/streptomycin, 2 % NeuroBrew21 with retinoic acid (Miltenyi Biotec), 2 µM PD0332991 (Selleckchem), 10 µM DAPT (Sigma-Aldrich), 0.6 mM CaCl2 (to give 1.8 mM CaCl2 in final complete medium (Sigma-Aldrich)), and 200 mM ascorbic acid (Sigma-Aldrich). The medium was replaced at day 7 post-plating by SynaptoJuice B medium which contained equal parts Advanced DMEM/F12 (with Glutamax) and Neurobasal A (Life Technologies), 1% penicillin/streptomycin (Life Technologies), 2% NeuroBrew-21 with retinoic acid (Miltenyi Biotec), 2 µM PD0332991 (Selleckchem), 3 µM CHIR 99021 (Tocris Bioscience), 0.3 mM CaCl2 (1.8 mM CaCl2 final), and 200 µM ascorbic acid (Sigma-Aldrich). Plated neurons were maintained for up to 14 days before exposure to TrkB ligands (see below). H9 ES cell-derived neurons were treated for 30 min, 2 h, 12 h and 24 h with BDNF (50 ng/ml), ZEB85  (5 μg/ml) and NT4 (75 ng/ml). Cultures were washed with D-PBS and lysed with 350 μl of RLT RNA lysis buffer containing 1 % β-mercaptoethanol. Total RNA was extracted from human neurons using the RNeasy Mini kit (Qiagen,). The TruSeq® Stranded mRNA Sample Preparation Guide and the high-throughput (HT) kit from Illumina was used according to the manufacturer's instructions.
attributes.tag: Experimental Factor: stimulus
attributes.value: brain-derived neurotrophic factor
attributes.tag: Experimental Factor: sampling time point
attributes.value: 0.5