Orangutan NPC 2
Pongo abelii
Protocols: Previously generated gorilla, orangutan and cynomolgus macaque wild-type and dCas9-KRAB iPS cell lines (Geuder et al. 2021, Kliesmete et al. 2023, Edenhofer at al. 2024) were cultured under feeder-free conditions on Geltrex (Thermo Fisher) -coated dishes in StemFit medium (Ajinomoto) supplemented with 100 ng/ml recombinant human basic FGF (Peprotech), 100 U/ml Penicillin and 100 μg/ml Streptomycin (Thermo Fisher) at 37 °C with 5% carbon dioxide. Two orangutan iPSC cell lines were differentiated into neural precursor cells (NPCs) via dual-SMAD inhibition as three-dimensional aggregation culture (Chambers et al. 2009, Ohnuki et al. 2014). Briefly, iPSCs were dissociated and 9000 iPSCs were seeded in a low attachment U-bottom 96-well-plate in 8GMK medium consisting of GMEM (Thermo Fisher), 8% KSR (Thermo Fisher), 5.5 ml 100× NEAA (Thermo Fisher), 100 mM Sodium Pyruvate (Thermo Fisher), 50 mM 2-Mercaptoethanol (Thermo Fisher) supplemented with 500 nM A-83–01 (Sigma Aldrich), 100 nM LDN 193189 (Sigma Aldrich) and 30 µM Y27632 (biozol). Culture medium of the spheres was changed every second day until they were harvested or plated for further culture. In order to obtain stable NPC lines, spheres were dissociated on day 7 of the differentiation process using Accumax (Sigma Aldrich) and plated onto Geltrex (Thermo Fisher) coated dishes. NPCs were subsequently cultured in NPC proliferation medium (DMEM F12 (Fisher Scientific) supplemented with 2 mM GlutaMAX-I (Fisher Scientific), 20 ng/mL bFGF (Peprotech), 20 ng/mL hEGF (Miltenyi Biotec), 2% B-27 supplement (50×) minus vitamin A (Gibco), 1% N2 supplement 100× (Gibco), 200 µM L-ascorbic acid 2-phosphate (Sigma), and 100U/ml 100µg/ml penicillin-streptomycin). The NPC lines were passaged 2-4 times before being dissociated and profiled using ATAC-seq together with the iPSC clones. After cells were counted, 100,000 cells were pelleted at 500 rcf for 5 min, washed with 1 ml PBS and pelleted at 500 rcf for 5 min at 4 °C. The supernatant was removed completely and cells were resuspended in 100 µl chilled nuclei lysis buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2 in water, supplemented with 0.1% Tween-20, 0.1% NP40, 0.01% Digitonin and 1% BSA) by pipetting up and down three times, followed by incubation on ice for 3 min. After lysis, 1 ml of lysis wash buffer (10 mM Tris-HCl pH7.4, 10 mM NaCl, 3 mM MgCl2 in water, supplemented with 0.1% Tween-20 and 1% BSA) was added, and tubes were inverted three times. After counting, 50,000 nuclei were pelleted at 500 rcf for 10 min at 4°C, the supernatant was removed and nuclei were resuspended in 50 µl transposition mix (25 µl 2x TD buffer, 2.5 µl TDE1, 16.5 µl PBS, 0.5 µl 1% digitonin, 0.5 µl 10% Tween-20 and 5 µl ddH2O) by pipetting six times. Transposition reactions were incubated at 37 °C for 1 h at 1000 rpm shaking, followed by a clean-up using the DNA Clean & Concentrator-5 kit (Zymo). For library generation, 20 µl of the transposed sample was mixed with 2.5 µl 25µl p5 custom primer, 2.5 µl 25µl p7 custom primer from Buenrostro et al. 2013 and 25 µl NEBNext Ultra II Q5 2x Master Mix (NEB) and a PCR with 10 cycles was conducted as stated in the Omni-ATAC protocol. Libraries were purified using the DNA Clean & Concentrator-5 kit, run on a 2% E-Gel (Thermo Fisher) and gel excision of DNA between 150 bp and 1,500 bp was performed using the Monarch DNA Gel Excision Kit (NEB).
ENA first public
2025-10-30
INSDC center name
LMU Munich, Faculty of Biology, Anthropology and Human Genomics
INSDC status
public
Submitter Id
E-MTAB-15654:Orangutan NPC 2
broker name
ArrayExpress
cell line
70Ab1
cell type
neural precursor cell
collection date
not collected
common name
Sumatran orangutan
genotype
wild type genotype
geographic location (country and/or sea)
not collected
individual
Jahe
isolate
not applicable
progenitor cell type
induced pluripotent stem cell
sample name
E-MTAB-15654:Orangutan NPC 2
scientific_name
Pongo abelii
sex
female