GSM7439128
Fresh tumor specimens were obtained from patients and processed for primary tumoroid culture (Wu and Nusse, Methods Mol Biol 2022). Tumoroids grown in Matrigel were harvested at P1 or P2 for single cell RNA sequencing. Briefly, dispase was added for 30 minutes at 37C with intermittent mechanical disruption of Matrigel drops. Tumoroids released from Matrigel were spun down at 50g for 5 minutes and washed twice with liver perfusion media/5% FBS. Tryple Express was added for 10-15 minutes at 37C to digest tumoroids to single cells. Liver perfusion media/5% FBS was added at the end of the digestion and cells were collected by centrifugation at 300g x 5 minutes. Cells were resuspended in William's media/10% FBS, counted, and resuspended in the appropriate volume to target 5000 cells for 10x scRNA sequencing. Libraries were constructed according to the manufacturer's specifications (10x Genomics 3' v2 for sample HB1 and v3 for all other samples)