GSM8262881: PDAC validation experiment 2, D7, row 1,cDNA; Homo sapiens; RNA-Seq
Patient-derived pancreatic cancer (PDAC) organoids were derived as previously described from patients with metastatic pancreatic cancer (Raghavan et al., Cell, 2021) and cultured in Growth factor Reduced Matrigel, fed with human complete organoid medium (Advanced DMEM/F12, 10 mM HEPES, 1x GlutaMAX, 500 nM A83-01, 50 ng/mL mEGF, 100 ng/mL mNoggin, 100 ng/mL hFGF10, 10 nM hGastrin I, 1.25 mM N-acetylcysteine, 10 mM Nicotinamide, 1x B27 supplement, RSPONDIN-1 conditioned media 10% final, WNT3A conditioned media 50% final, 100 U/mL penicillin/streptomycin, and 1x Primocin). Cells were seeded in seeded in 96-well plates and allowed to rest for 24 hours before ligand treatment where each well received 4 or 5 ligands in the compressed screen or only 1 ligand for the conventional validation screen. Assay plates were cultured for 7 days at 37°C/5% CO2 and processed for scRNA-seq with cell hashing. PDAC organoids for compressed screens and validation experiments were transferred to the upper chamber of a 96 well filter plate on a vacuum manifold and media was aspirated under vacuum, to only retain organoids larger than 30-40 µms. Additional media was added and aspirated under vacuum to remove residual Matrigel and cell debris. Organoids were dissociated into single cells by resuspending with TrypLE Express for 20 minutes at 37°C with intermittent mixing. RPMI 1640 with 50 µg/mL DNase was added to quench digestion, and the single cell suspension was collected by centrifugation into a 96-well u-bottom ultra-low attachment plate and Quenching solution was aspirated. Cell staining solution (45uL cell staining buffer + 5uL human multiplexing antibody from BD Biosciences) was added to each well and incubated at room temperature for 20 minutes. Human multiplexing antibodies were distributed column-wise with each column having a unique tag, and samples were collapsed row-wise (containing non-overlapping tags). Usinng Honeycomb Bio HIVEs, 15,000 cells were loaded into each hive and the library was constructed following the Honeycomb Bio HIVE protocol as described in their October 2021 protocol (v21.10), with modifications to accommodate cell hashing.