Illumina NovaSeq 6000 paired end sequencing; ATAC-seq of iPSC-derived NPCs and neurons
ATAC-seq of iPSC-derived NPCs and neurons
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.  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)
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 Endocrin
E-MTAB-9087:NPC10_rep2_p