NextSeq 500 sequencing; RNA-seq of human neurons derived from mutant embryonic stem cell line lacking the BDNF receptor TrkB treated with the natural TrkC receptor agonist Neurotrophin-3
RNA-seq of human neurons derived from mutant embryonic stem cell line lacking the BDNF receptor TrkB treated with the natural TrkC receptor agonist Neurotrophin-3
Cultures were washed with D-PBS and lysed with 350 μl of RLT RNA lysis buffer containing 1 % β-mercaptoethanol. Human ES cells lacking TrkB designated A10 were plated on matrigel-coated plates (Corning, VWR) and maintained in mTSER medium (STEMCELL Technologies). hES cells were passaged with RelesR from (StemCell Technologies) 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). 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 TrkC ligand. 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.