Single cell RNA sequencing of cultured CCR10+ and CCR10- lung airway epithelial cells

Mechanically dissociated IPF lung explant derived cellular suspensions were cultured in 50% senescent lung fibroblast conditioned medium + 50% fibroblast complete medium (DMEM + 15% FBS + antiboitics) and 10 µM of Y27632. After approximately 2 weeks, cells were passaged and cultured overnight in Pneumacult Ex Plus medium (STEMCELL technologies). Overall design: Explanted IPF lung tissues were mechanically minced to release loosely adherent and non-adherent cells. Cells were purified by passing the dissociated suspensions through a sterile 100 µm filter and then cultured in CRC medium: 50% senescent lung fibroblast conditioned medium + 50% fibroblast complete medium (DMEM + 15% FBS + antiboitics) and 10 µM of Y27632. Medium was changed every 3 days for approximately two weeks. After two weeks, cells were dissociated using Accutase solution and replated in PnemaCult Ex Plus medium (STEMCELL technologies) containing 10 µM Y27632 overnight. The next morning epithelial cells were dissociated using Accutase solution, stained with anti-EpCAM and -CCR10 antibodies and EpCAM+ CCR10+ and EpCAM+ CCR10- epithelial cells were FACS sorted using a BD Aria III sorter. Single sorted cells were then caputred and sequenced using 10x genomics platform.