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

ABSTRACT: 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.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Homo sapiens

SUBMITTER: Alice Pébay 

PROVIDER: E-MTAB-8511 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress