title: GSM4996503: In vitro hESC-RPE neuronal differentiation day 70; Homo sapiens; RNA-Seq
library_construction_protocol: Cell sorting was performed on hPSC-RPE cultures after 30 days of hESC-RPE differentiation. Samples were stained with BV421 Mouse Anti-Human CD140b (BD Biosciences 564124, clone [28D4], 10 μg/mL), BB515 Mouse Anti-Human CD56 (BD Biosciences 564489, clone [B159], 2.5 μg/mL), PE Mouse anti-human Ki-67 (Biolegend 350504, clone [Ki-67], 50 μg/mL) conjugated antibodies, diluted in 2% FBS and 1 mM EDTA (ThermoFisher Scientific, 10082147). Cells were incubated with the conjugated antibodies on ice for 30 min. Stained cells were then sorted using a BD FACS Aria Fusion Cell Sorter (BD Bioscience) using FACSDiva Software v8.0.1. Directly after sorting, 68,420 cells/cm2 were plated on matrigel coated plates. Cells were cultured until confluency in P1 media with DMEM/F12 (ThermoFisher Scientific, 11320033) as basal media containing B27 (ThermoFisher Scientific, 17504044), N2 (ThermoFisher Scientific, 17502048), 10ng/mL hDKK1 (R&D Systems, 5439-DK-010), 10ng/mL mouse Noggin (R&D Systems, 1967-NG-025), 10ng/mL hIGF-1 (R&D Systems, 291-G1-200), 5ng/mL bFGF (ThermoFisher Scientific, 13256029) and 50 U/mL of Penicillin-Streptomycin (ThermoFisher Scientific, 15140122). Thereafter and until day 40, cells were continued cultured in P1 with the addition of 40ng/mL 3,3′,5-Triiodo-L-thyronine T3 (Sigma, T-074-1ML) and 100uM Taurine (Sigma, T8691-25G). This protocol was based on previous work (Shao et al., 2017). Cells were transported at 4°C to the Eukaryotic Single Cell Genomics Facility (ESCG, SciLifeLab, Stockholm, Sweden) where a 3' cDNA library was prepared for single cell RNA sequencing (scRNA-seq) using the 10X Genomics platform and NovaSeq 6000 software.