title: Illumina NovaSeq 6000 paired end sequencing; ATAC-seq of human (Homo sapiens) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines
design: ATAC-seq of human (Homo sapiens) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines
library_construction_protocol: Previously generated urinary stem cell derived iPS-cells (Homo sapiens) of 2 human individuals and fibroblast derived cynomolgus macaque iPSCs (Macaca fascicularis) of 2 individuals (Geuder et al. 2021) were differentiated to neural progenitor cells 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).  All cell lines have been authenticated using RNA sequencing (RNA-seq) (Geuder et al. 2021), and the current study. iPSCs of 2 clones from 2 human individuals and 2 clones of 2 cynomolgus macaque individuals were differentiated using the protocol as described above. The NPC lines were cultured in NPC proliferation medium and passaged  2 - 4 times until they were dissociated and subjected to ATAC-seq together with the respective 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).