title: GSM4996491: In vitro hESC-RPE differentiation day 30; Homo sapiens; RNA-Seq
library_construction_protocol: A step-by-step protocol describing the differentiation procedure has been reported previously (Plaza Reyes et al., 2020a, 2020b). hESCs were plated at a cell density of 2.4x104 cells/cm2 on hrLN-521 (20 μg/mL) using NutriStem hPSC XF medium. A Rho-kinase inhibitor (Millipore, Y-27632) at a concentration of 10 μM was added during the first 24h, while cells were kept at 37°C, 5% CO2/5% O2. After 24h, hPSC medium was replaced with differentiation medium NutriStem hPSC XF without bFGF and TGFβ and cells were placed at 37°C, 5% CO2/21%O2. From day 6 after plating, 100 ng/mL of Activin A (R&D Systems, 338-AC-050) was added to the media. Cells were fed three times a week and kept for 30 days. Monolayers were then trypsinized using TrypLE Select (ThermoFisher Scientific, 12563011) for 10 min at 37°C, 5% CO2. The enzyme was carefully removed and the cells were collected in fresh pre-warmed NutriStem hPSC XF medium without bFGF and TGFβ by gentle pipetting to obtain a single cell suspension. Cells were centrifuged at 300 g for 4 min, the pellet was resuspended, passed through a cell strainer (ø 40 μm, VWR, 732-2757) and seeded on laminin coated dishes (hrLN-521 at 20μg/mL) at different cell densities ranging from 1.4x106 to 1.4x104 cells/cm2. Replated cells were fed three times a week during the subsequent 30 days with NutriStem hPSC XF medium without bFGF and TGFβ. For hPSC-RPE in vitro differentiation in 3D suspension embryoid bodies see our previous publication (Plaza Reyes et al., 2016). 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.