Illumina NovaSeq 6000 paired end sequencing; Promoter Capture-C of iPSC-derived NPCs and neurons
Promoter Capture-C of iPSC-derived NPCs and neurons
Cells (6 technical replicates from CHOPWT10 and CHOPWT14 neurons, 3 replicates from CHOPWT10 and CHOPWT14 NPCs) were fixed with formaldehyde on plate for 10 min, and then were neutralized with glycine (final concentration is 125mM). The fixed cells were scraped together with media, centrifuged to remove the fixation solution. The cell pellets were then washed with PBS and frozen under -20 oC. 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. 10 million fixed cells were thawed at 37ºC, followed by centrifugation at RT for 5 mins at 14,000rpm. The cell pellet was resuspended in 1 mL of dH2O supplemented with 5 uL 200X protease inhibitor cocktail, incubated on ice for 10 mins, then centrifuged. The cell pellet was resuspended to a total volume of 650 uL in dH2O.  50 uL of cell suspension was set aside for pre-digestion QC, and the remaining sample was divided into 6 tubes. Both pre-digestion controls and samples underwent a pre-digestion incubation in a Thermomixer (BenchMark) with the addition of 0.3%SDS, 1x NEB DpnII restriction buffer, and dH2O for 1hr at 37ºC shaking at 1,000rpm. A 1.7% solution of Triton X-100 was added to each tube and shaking was continued for another hour. After the pre-digestion incubation, 10 ul of DpnII (NEB, 50 U/µL) was added to each sample tube only, and continued shaking along with pre-digestion control until the end of the day. An additional 10 µL of DpnII was added to each digestion reaction and digested overnight.  The next day, a further 10 µL DpnII was added and continue shaking for another 2-3 hours. 100 uL of each digestion reaction was then removed, pooled into one 1.5 mL tube, and set aside for digestion efficiency QC.  The remaining samples were heat inactivated incubated at 1000 rpm in a MultiTherm for 20 min, at 65°C to inactivate the DpnII, and cooled on ice for 20 additional minutes. Digested samples were ligated with 8 uL of T4 DNA ligase (HC ThermoFisher, 30 U/µL). and 1X ligase buffer at 1,000 rpm overnight at 16°C in a MultiTherm. The next day, an additional 2 µL of T4 DNA ligase was spiked in to each sample and incubated for another few hours. The ligated samples were then de-crosslinked overnight at 65°C with Proteinase K (20 mg/mL, Denville Scientific) along with pre-digestion and digestion control. The following morning, both controls and ligated samples were incubated for 30 min at 37°C with RNase A (Millipore), followed by phenol/chloroform extraction, ethanol precipitation at -20°C, then the 3C libraries were centrifuged at 3000 rpm for 45 min at 4°C to pellet the samples Isolated DNA from 3C libraries was quantified using a Qubit fluorometer (Life technologies), and 10 μg of each library was sheared in dH2O using a QSonica Q800R to an average fragment size of 350bp.  QSonica settings used were 60% amplitude, 30s on, 30s off, 2 min intervals, for a total of 5 intervals at 4 °C. After shearing, DNA was purified using AMPureXP beads (Agencourt). DNA size was assessed on a Bioanalyzer 2100 using a DNA 1000 Chip (Agilent) and DNA concentration was checked via Qubit. SureSelect XT library prep kits (Agilent) were used to repair DNA ends and for adaptor ligation following the manufacturer protocol. Excess adaptors were removed using AMPureXP beads. Size and concentration were checked again by Bioanalyzer 2100 using a DNA 1000 Chip and by Qubit fluorometer before hybridization. One microgram of adaptor-ligated library was used as input for the SureSelect XT capture kit using manufacturer protocol and our custom-designed 41K promoter Capture-C probe set. The quantity and quality of the captured libraries were assessed by Bioanalyzer using a high sensitivity DNA Chip and by Qubit fluorometer.
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-9159:neuron_CHOPWT10_rep3_p