scRNA seq of the hESC line H9 differentiated to retinal pigment epithelium at two different time points

We differentiated the human embryonic stem cell line H9 into retinal pigment epithelium (RPE) cells, to assess their transcriptomic profiles over time in culture. In-depth molecular analysis was performed by single cell RNA-Sequencing to access the molecular signature of RPE cells grown and harvested at two time points in culture (30 days and 369 days post passaging). We performed high-resolution comparisons at subpopulation and single-cell levels, to assess gene expression pathway signatures in RPE cells and upon aging in vitro. The hESC line H9 was grown to 70-80% confluence, transitioned to E6 medium for 2 days, with supplementation of N2 from day 2 until day 33. On day 33, medium was switched to RPEM (alpha-MEM, N1 supplement, 5% FBS, NEAA, Pen Strep Glutamine, taurine-hydrocortisone-triiodo-thyronin (THT)) for an additional 32 days. Cells were enzymatically passaged using 0.25% Trypsin EDTA and plated at 75,000 cells/cm2 on Matrigel growth factor reduced pre-coated plates (P1). Cells were subsequently harvested at day 30 (sample \"YOUNG\") and day 369 (sample \"AGED\"). Both samples were sorted for live cells using PI on a BD FACS Aria. Cells were centrifuged at 300g for 5 min and resuspended in PBS containing 0.04% BSA to a concentration of ~800-1000 cells/ µl. Approximately 17,400 cells were loaded onto a 10X chip single Cell 3' Chips along with the reverse transcription master mix as per the manufacturer's protocol for the Chromium Single Cell 3' v2 Library for a target recovery of 10,000 cells. Samples were then processed for sc Seq.