GSM7429314: Inner ear organoid d40 SOX2nT+; Homo sapiens; RNA-Seq
D20 and d30 aggregates were washed with 0.5 mM EDTA in DPBS, followed by incubation with 1.1 mM EDTA and TrypLE in DPBS at 37°C for 60 min. While incubation, the samples were mechanically dissociated on low-cell-binding 24-well plates and gently pipetted with P1000 tips. After confirming most SOX2ntdTomato-positive cells become single cells, they were filtered sequentially through a 100-µm and a 40-µm cell strainer and then transferred into 2-ml tubes. After spinning down at 100 × g for 5 min, resuspend the cells with fluorescence-activated cell sorting (FACS) buffer which consists of 2% fetal bovine serum in 1X DPBS and collected into a 5-ml round-bottom polypropylene tube . D40, d50, and d60 aggregates were dissected to remove cartilage-like tissues and obvious non-epithelial tissues in OMM in a 60-mm dish with Style 5 tweezers under a fluorescence stereomicroscope. The dissected aggregates were kept in the incubator in OMM on low-cell-binding 6-well plates until dissociation. After dissociation with Accutase, SOX2ntdTomato-positive populations were collected with BD SORP Aria or BD FACSAria Fusion. Single-cell suspension after FACS was centrifuged and resuspended with FACS buffer. The single-cell gene expression analysis was conducted using a 10X Chromium single-cell system (10X Genomics). Approximately 10,000 cells per sample were loaded on a single cell master mix with lysis buffer and reverse transcription reagents, according to the Chromium Single Cell 3' Reagent Kits V3 User Guide, CG000183 Rev A (10X Genomics). Along with the single-cell gel beads and partitioning oil, the single-cell master mixture containing the single-cell suspension was dispensed onto a Single Cell Chip B in separate wells, and the chip was loaded to the Chromium Controller for gel bead-in emulsion (GEM) generation and barcoding, followed by cDNA synthesis and library preparation. At each step, the quality of cDNA and library was examined by Bioanalyzer and Qubit. The resulting library was sequenced in a custom program for 28b plus 91b paired-end sequencing on an Illumina NovaSeq 6000 sequencer. Over 27K reads per cell were generated and 93% of the sequencing reads reached Q30 (99.9% base call accuracy).?