GSM6731130: BLCa77 organoids; Homo sapiens; RNA-Seq
Organoids were collected in Basis medium (100g, 5min) and dissociated into single cells with 1ml TrypLE Express at 37°C for 10 min. Single-cell suspension was counted and cryopreserved in FBS/10%DMSO before scRNA sequencing. To obtain single cells from tumor tissue, tissues were digested as explained in the protocol for organoid derivation, and then cells were counted and cryopreserved in FBS/10%DMSO before analysis.  Slow-frozen single cell suspensions were shipped to the Genomics Facility Basel for scRNA-seq processing. Cryovials were thawed and content transferred into 10 mL Basis medium and incubated for 10 min at room temperature to ensure complete removal of DMSO from the cells. Cells were collected (350g, 5 min), resuspended in 1 mL washing buffer consisting of PBS (Ca2+/Mg2+-free, Gibco, 10010-015) and 1% BSA (Sigma-Aldrich, A7906) and sequentially filtered through 100 µm (Falcon, 352360) and 40 µm cell strainers (Falcon, 352340). Apoptotic cells were removed by immunomagnetic cell separation using the Annexin Dead Cell Removal Kit (StemCell Technologies, 17899) and EasySepTM Magnet (StemCell Technologies, 18000). Whenever possible and required, repeat dead cell removal was performed to increase cell viability. Next, cells were washed with a resuspension buffer (PBS with 0.05% BSA), spin down and resuspended in a resuspension buffer. Cell numbers and viability were assessed at each step on a Cellometer K2 Image Cytometer (Nexcelom Bioscience, Cellometer K2) using ViaStain AOPI Staining Solution (Nexcelom Bioscience, CS2-0106-5mL) and PD100 cell counting slides (Nexcelom Bioscience, CHT4-PD100-003). Optimal cell concentrations were set according to 10x Genomics protocols (700-1200 cells/µL). Cells were loaded and processed using the 10x Genomics Chromium platform with the Next GEM Single Cell 3' Reagent Kit v3.1 on 10x Genomics Chromium Single Cell Controller (10x Genomics, PN-120263). 4'000 cells were targeted per sample. Gene expression (GEX) libraries were amplified, pooled and sequenced on the Illumina NovaSeq 6000 platform with addition of 1% PhiX at recommended sequencing depth (20,000-50,000 reads/cell).