biostudies-arrayexpressAnita TérmegMacaca fascicularisbwa-mem2, samtools, Genrichhttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15654To compare chromatin accessibility across three primate species, between wild-type (WT) and genetically modified induced pluripotent stem cell (iPSC) lines, and between the iPSC state and neural precursor cells (NPCs) derived from these iPSCs, we generated ATAC-seq data from nine primate samples. The samples included two gorilla WT iPSC samples and one gorilla KRAB-dCas9 iPSC sample (all from the same individual), one orangutan WT iPSC sample, one orangutan KRAB-dCas9 iPSC sample and two orangutan NPC samples (from two different individuals), and one cynomolgus macaque WT iPSC sample and one cynomolgus macaque KRAB-dCas9 iPSC sample (from the same individual). The gorilla and orangutan iPSCs were derived from urinary stem cells (Geuder et al. 2021), while the cynomolgus macaque iPSCs were derived from skin-fibroblasts. The KRAB-dCas9 iPS cell lines were created by stably integrating dox-inducible KRAB-dCas9-HA-P2A-mCherry construct at the AAVS1 locus (Edenhofer et al. 2024). NPCs were obtained by the directed differentiation of iPSCs via dual-SMAD inhibition (Chambers et al. 2009; Ohnuki et al. 2014). ATAC-seq libraries were generated using the Omni-ATAC protocol (Corces et al. 2017) with minor modifications.biostudies-arrayexpressNucleic Acid Extraction - 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).Sequencing - Libraries were pooled and sequenced on a NextSeq 1000 instrument with the following setup: R1: 110, i7: 8, i5: 8, R2: 110.Library Construction - 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).Sample Collection - 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.OrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Gorilla, orangutan and cynomolgus macaque reads were mapped to the gorGor6, ponAbe3 and macFas6 reference genomes, respectively. For mapping, we used bwa-mem2 (Vasimuddin et al. 2019) with the parameters -M -t 8 -I 250,150. BAM files were name sorted using samtools (Li et al. 2009), and then peaks were called using Genrich (https://github.com/jsh58/Genrich). The peak calling was done per species, cell type and genotype with the parameters -j -y -r -q 0.05 -a 200 -e MT,Y -s 20, without the use of a blacklist.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsNextSeq 1000ATAC-seqMacaca fascicularisAnita TérmegfalseATAC-seq of gorilla (Gorilla gorilla), orangutan (Pongo abelii) and cynomolgus macaque (Macaca fascicularis) iPSC, KRAB-dCas9 iPSC and iPSC-derived NPC cell linesTo compare chromatin accessibility across three primate species, between wild-type (WT) and genetically modified induced pluripotent stem cell (iPSC) lines, and between the iPSC state and neural precursor cells (NPCs) derived from these iPSCs, we generated ATAC-seq data from nine primate samples. The samples included two gorilla WT iPSC samples and one gorilla KRAB-dCas9 iPSC sample (all from the same individual), one orangutan WT iPSC sample, one orangutan KRAB-dCas9 iPSC sample and two orangutan NPC samples (from two different individuals), and one cynomolgus macaque WT iPSC sample and one cynomolgus macaque KRAB-dCas9 iPSC sample (from the same individual). The gorilla and orangutan iPSCs were derived from urinary stem cells (Geuder et al. 2021), while the cynomolgus macaque iPSCs were derived from skin-fibroblasts. The KRAB-dCas9 iPS cell lines were created by stably integrating dox-inducible KRAB-dCas9-HA-P2A-mCherry construct at the AAVS1 locus (Edenhofer et al. 2024). NPCs were obtained by the directed differentiation of iPSCs via dual-SMAD inhibition (Chambers et al. 2009; Ohnuki et al. 2014). ATAC-seq libraries were generated using the Omni-ATAC protocol (Corces et al. 2017) with minor modifications.2025-10-30T00:00:00Z2026-05-26T16:03:01.784Z2025-10-02T21:35:31.754ZE-MTAB-15654ERP180957E-MTAB-13373EFO_0002944EFO_0007045EFO_0004170EFO_0005518EFO_0003816EFO_0004184