GSM9309247: EPCAM- in d109 cochlear organoids without T4; Homo sapiens; RNA-Seq
Aggregates were dissected to enrich the otic epithelial cell types 2-3 days prior to single-cell dissociation. On the day of cell sorting, aggregates were incubated with Accumax at 37°C for 120 min. During incubation, the samples were mechanically dissociated by gentle pipetting with wide-orifice P1000 tips. After confirming that most cells, especially those with reporter expression, were completely dissociated, they were filtered through a 40-μm cell strainer and then transferred into 2-ml tubes. After spinning down at 200 ×g for 5 min, dissociated cells were resuspended in FACS buffer consisting of 2% fetal bovine serum in 1× DPBS. Then, cells were stained with 1:100 PE conjugated EPCAM antibody at 4℃ for 30 min in dark. After antibody staining, cells were washed and resuspended in FACS buffer. Cells were stored on ice in the dark prior to sorting. Both PE-EPCAM+ and PE-EPCAM- populations were collected in separate tubes for separate downstream scRNA-seq reactions. scRNA-seq for all samples was performed using the 10x Genomics Chromium 3' v3 platform for cDNA library construction and the NovaSeq 6000 system for sequencing.