description: Protocols: Five replicates of CHOPWT10 and CHOPWT14 NPCs or CHOPWT10 and CHOPWT14 neurons were harvested using Accutase, followed by a DPBS wash, then counted. 50,000 cells of each sample were spun down at 550 ×g for 5 min at 4°C. The cell pellet was then resuspended in 50 μl cold lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630) and spun down immediately at 550 ×g for 10 min, 4°C. 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. The nuclei were resuspended on ice in the transposition reaction mix (2x TD Buffer, 2.5ul Tn5 Transposase and Nuclease Free H2O) (Illumina Cat #FC-121-1030, Nextera) on ice and the transposition reaction was incubated at 37°C for 45 min. The transposed DNA was then purified using a the MinElute Kit (Qiagen) adjusted to 10.5 μl elution buffer. The transposed DNA was converted into libraries using NEBNext High Fidelity 2x PCR Master Mix (NEB) and the Nextera Index Kit (illumina) by PCR amplification for 12 cycles. The PCR reaction was subsequently cleaned up using AMPureXP beads (Agencourt), checked on a Bioanalyzer 2100 (Agilent) high sensitivity DNA Chip (Aglient)
attributes.tag: ENA first public
attributes.value: 2021-05-01
attributes.tag: ENA last update
attributes.value: 2021-05-27
attributes.tag: External Id
attributes.value: SAMEA6847268
attributes.tag: INSDC center alias
attributes.value: Center for Spatial and Functional Genomics, Children's Hospital of Philadelphia, Philadelphia, 19104, PA, USA Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, 19104, PA, USA Divisions of Genetics and Endocrinology, Children's Hospital of Philadelphia, Philadelphia, 19104, PA, USA
attributes.tag: INSDC center name
attributes.value: Center for Spatial and Functional Genomics, Children's Hospital of Philadelphia, Philadelphia, 19104, PA, USA Department of Pediatrics, University of Pennsylvania Perelman School of Medicine, Philadelphia, 19104, PA, USA Divisions of Genetics and Endocrinology, Children's Hospital of Philadelphia, Philadelphia, 19104, PA, USA
attributes.tag: INSDC first public
attributes.value: 2021-05-01T00:23:33Z
attributes.tag: INSDC last update
attributes.value: 2021-05-27T04:10:04Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-9087:Neur10_rep3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: CHOPWT10
attributes.tag: cell type
attributes.value: neuron
attributes.tag: common name
attributes.value: human
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: normal
attributes.tag: genotype
attributes.value: wild type genotype
attributes.tag: organism part
attributes.value: blood
attributes.tag: progenitor cell type
attributes.value: induced pluripotent stem cell
attributes.tag: sample name
attributes.value: E-MTAB-9087:Neur10_rep3