GSM6560704: Tx-iPSC-RPE_2; Homo sapiens; RNA-Seq
For in vitro RPE cultures, day 90 ESC-RPEs (n=1) and iPSC-RPEs (n=1) grown on Transwell inserts were dissociated into single cells with TrypLE Express for 30 min, at 37°C, 5% CO2 and resuspended to 1,000 cells/μL in 0.04% BSA in PBS prior to scRNA-seq. Six animals (Tx-ESC-RPE: n=3; Tx-iPSC-RPE: n=3) were sacrificed in deep anesthesia at 4-weeks post-surgery. After enucleation, the entire globes were washed thoroughly with PBS. The anterior segments were removed, and posterior poles (approximately 5 x 5 mm) were cut from the globes. The neuroretina was carefully removed, and the implant was taken out, and transferred into an Eppendorf tube with 500 μL PBS, using intraocular forceps (Alcon). The cells on implant were dissociated with 0.25% Trypsin-EDTA (Gibco) for 20 to 30 min at 37 °C, 5% CO2 with gentle resuspending every 5 min. Tx-RPE cells were filtered using a 30 µm strainer and resuspended in 0.04% BSA in PBS. RPE cell counting was performed. Due to the low number of cells retrieved, we spiked-in A549 (CCL-185, ATCC) immortalized cells to avoid library preparation failure due to insufficient cell count. The concentration was adjusted to 1,000 cells/μL in 0.04% BSA in PBS prior to scRNA-Seq. Single cell suspensions from in vitro ESC- and iPSC-RPE and from transplanted Tx-ESC- and Tx-iPSC-RPE were maintained at 4 °C for less than 30 min before being transported to 10x Genomics Chromium instrument for  scRNA-seq. Using the 10x Genomics Single Cell 3' Reagent Dual Index Kit V3, we performed droplet based single cell encapsulation, followed by bar-coded reverse transcription to generate the bar-coded complementary DNA (cDNA) library.