GSM5140930: CCOs differentiation, day 8; Homo sapiens; RNA-Seq
Organoids of various ages (Day 8, Day 14, and Day 21) were dissociated using TrypLE (ThermoFisher, 12604013). Organoids were washed once with 1xPBS and then incubated in TrypLE solution at 37 °C for 10 to 15 mins. Subsequently, organoids were dissociated into single cells by gentle pipetting using wide-bore tips. Cell solutions were then diluted with 1xPBS and passed through 40um strainer (Falcon, 352340) to remove any undissociated cell clumps. Cells were then centrifuged down to remove TrypLE and washed once with 1xPBS (300 × g for 5 min at 4 °C). Pellets were eventually re-suspended in 1xPBS and stored on ice. Cell viability and concentration were checked using an automated cell counter (BioRad). Cell stocks were diluted to around 1000 cells/uL with 1xPBS before downstream steps. Chromium™ Single Cell 3' Library & Gel Bead Kit v2 was used to construct the library.  For all library preparation, recovery of 6000 cells was aimed. Briefly, cells together with Gel Bead-in-Emulsions (GEM)-reverse transcription reagents, and GEM beads were loaded to the 10xChip, in which individual cells are captured, lysed, reverse transcribed and barcoded. Subsequently, barcoded cDNA was amplified and used for library construction. Libraries which passed the quality control were then sent out for sequencing. 10x Genomics single-cell RNA Library preparation and sequencing