GSM8859515: POU4F3nT+ in d140 cochlear organoids with T4; Homo sapiens; RNA-Seq
Aggregates were dissected to enrich POU4F3nT+ cells 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 collected into a 5-ml round-bottom polypropylene tube. Simultaneously, the above protocol prepared size control cells from wild-type WA25 hESCs. Cells were stored on ice in the dark prior to sorting. tdTomato-positive populations were collected and cleaned using a SORP Aria or SORP Fusion (BD Bioscience). scRNA-seq for all samples was performed using the 10x Genomics Chromium 3' v3 platform for cDNA library construction and the NovaSeq 6000 system (Illumina) for sequencing.