GSM9021482: GB cells pooled with associated Neural-like organoid; Homo sapiens; RNA-Seq
GB-FUCCI (FUCCI_GBM1) cells were enzymatically dissociated from adherent cultures using TrypLE™ Express Enzyme (Thermo Fisher Scientific, Cat# 12604013). Cell suspensions were centrifuged at 300 × g and resuspended in NB medium. Samples were fixed using the Evercode™ Cell Fixation v2 kit (Parse Biosciences, ECF2001/ECF2101) following the manufacturer's instructions. Fixed cells were stored at –80 °C for up to 1 month prior to barcoding. GB spheroids were co-cultured with either neural-like organoids, or foregut-like organoids. A few days were given for the GB cells to invade into the neigbouring organoid/ environment. The now “assembloid” was dissociated and the GB cells and environment cells separated by Fluorescence-Activated Cell Sorting through the means of GFP expressing GB cells. The cells were then pooled again at a 1:1 ratio. Library preparation was performed for the GB-FUCCI cells according to manufacturer instructions using the EvercodeTM WT Mini v2 kit (Parse Biosciences, ECW02010/ECW02110). Fixed single-cell suspensions underwent a series of intracellular, instrument-free reverse transcription barcoding reactions. Resulting barcoded libraries were divided into sublibraries as required, with an additional sublibrary-specific barcode introduced. Final sublibraries were lysed, and barcoded cDNA was amplified by PCR and prepared for Illumina sequencing. Satisfactory sublibrary quality control was checked and confirmed on a Tapestation 4200 (Agilent). Sequencing was performed using the NovaSeq 6000 platform with a resultant 100% clean reads.Library preparation for the pooled assembloid cells was performed using the 10x Genomics GEM-X 3′ v4 assay, and the libraries were processed and sequenced on an Illumina NovaSeq X Plus at 50,000 reads per cell using the 10x Genomics 3′ Single Cell Gene Expression Service provided by Single Cell Discoveries.