title: scRNAseq of gastric tumors and adjacent mucosa from Twist2Cre;Lkb1fl/+;Rosa26RTdtomato/+ mice
design: scRNAseq of gastric tumors and adjacent mucosa from Twist2Cre;Lkb1fl/+;Rosa26RTdtomato/+ mice
library_construction_protocol: Antral polyps and adjacent antral mucosa from Twist2Cre:Lkb1fl/+;Tdtomato+/- mice were dissociated into single cells as follows: the tissue was flushed with PBS, minced into < 1 mm pieces with scissors, and placed in a 15 ml falcon tube filled with 5 ml of a collagenase mixed solution (collagenase II (Gibco 17101-019) + IV (ThermoFischer 17104019), 1:1 ratio at 2 mg/ml). The samples were incubated horizontally in a 37°C shaking incubator (220 rpm) for 40 min. During this incubation time, pieces were pipetted up and down 10 times every 10 min, and the collagenase solution was changed after 20 minutes, collecting the supernatant containing the single cells. The supernatant (~10 ml) was then transferred to a clean Falcon tube, and collagenases were inactivated with cold DMEM supplemented with 20% fetal bovine serum (FBS). The samples were passed through a 70 um cell strainer, centrifuged for 10 min at 1100 rpm, and single cells were resuspended in 1–2 ml of PBS. Single-cell suspensions were stained with 7 AAD (LifeTechnologies #A1310) at a 1:1000 dilution prior to FACS sorting to exclude dead cells. 10.000 live cells per sample were sorted at ultra-purity using a Sony SH800Z Cell Sorter. The 10X v3 library kit (Library Construction Kit from 10X Genomics #1000190) was used for scRNA-seq library preparation.  The 10X v3 library kit (Library Construction Kit from 10X Genomics #1000190) was used for scRNA-seq library preparation.