HiSeq X Ten paired end sequencing; Transcriptional networks identify synaptotagmin-like 3 as a regulator of cortical neuronal migration during early neurodevelopment
Transcriptional networks identify synaptotagmin-like 3 as a regulator of cortical neuronal migration during early neurodevelopment
Human H9 ESCs (line WA09 (WiCell), passages 20–40) were maintained on a feeder layer of irradiated embryonic mouse fibroblasts (MEFs) in hESC medium consisting of DMEM/F12 (Life Technologies), 1×NEAA (Life Technologies), 0.5× GlutaMAX (Life Technologies), and 0.1 mM β-mercaptoethanol (Sigma). To feed the cells, 4 ng/ml FGF-2 (R&D Systems) was added, as described previously (Zhang et al., 2001). The hESCs were electroporated as previous research (Chen et al., 2015). Briefly, Rho-kinase (ROCK) inhibitor (0.5 μM, Calbiochem, H-1152P)-pretreated hESCs were digested, dispersed into single cells, and electroporated with 15 μg of Cas9 plasmid, 15 μg of sgRNAT2 plasmid, and 30 μg of donor plasmid in 500 μL of electroporation buffer (5 mM KCl, 5 mM MgCl2, 15 mM HEPES, 102.94 mM Na2HPO4, and 47.06 mM NaH2PO4 [pH 7.2]) using the Gene PulserXcell System (Bio-Rad) at 250 V and 500 μF in a 0.4-cm cuvette (Phenix Research Products).                               Forebrain cortical neurons were generated as described in earlier research (Zhang et al., 2001). In brief, Ctrl (hESC) or SYTL3-KO hESC lines on MEF were cultured in hESCs medium for 5–7days. Cells were digested by dispase for 2min and clones were collected by pipet and cultured in ES medium to form embryonic bodies. They were then induced to neuroepithelium in a neural induction medium consisting of DMEM/F12, 1×N2, and 1×NEAA containing SB431542 (Stemgent, 2μM) and DMH1 (Tocris, 2μM).Neural progenitors were collected at around 20 days after neural differentiation. For neuronal differentiation and maturation, neural progenitor clusters were dissociated with accutase (1 unit/ml, Invitrogen) at 37°C for 5min and placed onto Matrigel-coated coverslips in neurobasal medium for more than one week in the presence of trophic factors, including brain-derived neurotrophic factor (BDNF, 20ng/ml), glial-derived neurotrophic factor(GDNF,10ng/ml), insulin-like growth factor 1(IGF1, 10ng/ml), cAMP (1μM), and Compound E (all from R&D Systems).                                                     SYTL3 was knockout in hESCs by Crispr/Cas9 to generate SYTL3-KO cell line  RNA was harvested using Trizol reagent. NEB Next® UltraTM RNA Library Prep Kit for Illumina® （NEB #E7770） was used with 1 ug of total RNA each sample for the construction of sequencing libraries.  RNA sequencing using TruSeq mRNA library kit.