title: Illumina NovaSeq 6000 paired end sequencing; RNA-seq of iPSC-derived NPCs and neurons from CHOPWT10 and CHOPWT14 cell lines
design: RNA-seq of iPSC-derived NPCs and neurons from CHOPWT10 and CHOPWT14 cell lines
library_construction_protocol: Three replicates of CHOPWT10 and CHOPWT14 NPCs or CHOPWT10 and CHOPWT14 neurons were collected by Trizol Reagent (Invitrogen). Frozen NPCs from donor CHOPWT10 and CHOPWT14 were obtained from CHOP stem cell core and thawed slowly in 37°C water bath. The thawed cells were gently washed in Neural Expansion Media (49% Neurobasal Media (ThermoFisher, cat# 21103049), 49% Advanced DMEM/F12 (ThermoFisher, cat# 12634010) and 2% 50X Neural Induction Supplement) in a 15 ml conical tube, followed by centrifuging at 300x g for 5 mins. Cells were resuspended in 1 ml pre-warmed Neural Expansion Media with Rock inhibitor (Y-27632 compound, Stem Cell Technologies, cat# 72304) at a final concentration of 10 μM and a cell count performed. NPCs were seeded at a density of 150k cells/cm2 onto hESC-qualified Matrigel-coated plates (Corning, cat# 354277) in 2.5 ml/well Neural Expansion Media and cultured at 37°C in a humidified cell culture incubator with 5% CO2. The day after, the medium was changed to remove the Y-27632 compound. NPCs were expanded for 6-7 days in 2.5 ml Neural Expansion Media exchanged every 48 hours before harvesting.  To differentiate NPCs into cortical neurons, we followed the protocol from Jones et al (2017) and started by seeding expanded NPCs onto plates coated with hESC-qualified Matrigel. The NPCs were supplemented with 1:1 mixture of N2:B27 media (250 ml N2 media: 240 ml DMEM/F12 with Glutamax (ThermoFisher, cat#10565-018), 2.5 ml 100X N2 supplement (ThermoFisher, cat#17502048), 2.5 ml 200mM L-glutamine (Corning,cat#MT25-005-CI), 2.5 ml 100X MEM Non-Essential Amino Acids solution (ThermoFisher, cat#11140076), 2.5ul 14.3M beta-mercaptoethanol (Sigma-Aldrich, cat#M3148), 2.5ml 100X Antibiotic-Antimycotic (ThermoFisher, cat#152440112), 0.25ml 100mg/ml insulin (Sigma-Aldrich, cat#I0516);  250 ml B27 media: 240ml Neurobasal media (ThermoFisher, cat# 21103049), 5ml 50x B27 supplement (ThermoFisher, cat#17504044)). The terminally differentiated neurons were maintained in N2:B27 medium for 30-40 days, with replacing half of the media every other day before harvesting. RNA was then purified using the Directzol RNA Miniprep Kit (Zymol) and depleted of contaminating genomic DNA using DNAse I RNA samples were depleted of rRNA using the QIAseq FastSelect RNA Removal Kit then processed into libraries using the NEBNext Ultra Directional RNA Library Prep Kit for Illumina (NEB) according to manufacturer's instructions.
attributes.tag: Experimental Factor: cell type
attributes.value: neuron
attributes.tag: Experimental Factor: cell line
attributes.value: CHOPWT10