Illumina HiSeq 1500 sequencing; RNA-seq of of human (Homo sapiens), gorilla (Gorilla gorilla) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines
RNA-seq of of human (Homo sapiens), gorilla (Gorilla gorilla) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines
Previously generated urinary stem cell derived iPS-cells of human (Homo sapiens), gorilla (Gorilla gorilla) and fibroblast derived cynomolgus macaque iPSCs (Macaca fascicularis) (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.  Samples for RNA-seq were taken from 3 clones of 3 human individuals and 4 clones of 2 cynomolgus macaque individuals at the iPSC stage (time point 0) and after 1, 5, 7 and 9 days during the neural maturation process. Spheres were dissociated at each time point using Accumax (Sigma Aldrich) and live cells were sorted using the BD FACS Aria II.  cDNA libraries for samples from the different species and differentiation time points were generated using the prime-seq protocol (Janjic et al. 2022).