Single cell RNA-seq of of human (Homo sapiens), gorilla (Gorilla gorilla) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines
Single cell RNA-seq of of human (Homo sapiens), gorilla (Gorilla gorilla) and cynomolgus macaque (Macaca fascicularis) iPSC and iPSC-derived NPC cell lines
Primate iPSCs were cultured in StemFit + bFGF as described previously (Geuder et al., 2021). For differentiation, the cells were dissociated and 9 × 103 cells were plated into each well of 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). Medium was changed every second day.  Spheres were collected in a reaction tube, washed with PBS and fixed with 4% PFA. After the spheres were incubated in 10%, 20% and 30% sucrose solution, they were stored at -80°C until further processing. Embedded spheres were then cut using a cryostat to slices a 20 µm. For staining the slides were thawed, rinsed with PBSand incubated with 0.5% TritionX100 for one hour. Primary antibodies in blocking solution were subseqently incubated overnight at 4°C. After washing with 0.05 % TritonX100/PBS at 37°C for 30 minutes, secondary antibody and DAPI were added and incubated overnight. After another 0.05 % TritonX100/PBS treatment at 37°C for 30 minutes, slides were mounted using vectashield and imaged on a confocal microscope.  On day 0,1,3,5,7 and 9 cells were dissociated using accumax and single cell sorted using a BD FACS Aria II. The cells were sorted into 96-well plates containing lysis-buffer consisting of Phusion buffer, proteinase K and barcoded oligo-dT primers. Libraries were prepared using mcSCRB-seq (Bagnoli et al., 2018). The method was followed exactly as outlined in the step-by-step protocol (Bagnoli et al., 2018) with the exception of using 17 cycles for the pre-amplification.