title: GSM8791775: ApcMin 1d after 2 Gy IR biol rep 1, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Macroscopic ApcMin/+ adenomas from all different anatomical regions of the small intestine were dissected and stored in high-glucose DMEM (Gibco, #11960-044) medium on ice. After isolation of all adenomas, the medium was replaced with a cell dissociation buffer containing 4 mg/ml dispase II (Sigma, #04942078001), 1 mg/ml collagenase I (Worthington, #LS004196), 1 mg/ml collagenase H (Roche, #11074032001), and 1000 U/ml benzonase (ChemCruz, #sc-202391) in HBSS (Gibco, #14175-053). After 30 min of incubation at 37°C, trypsin-EDTA (Thermo Fisher Scientific, #25200056) was added, and incubation was continued for 10 min at 37°C. The samples were vortexed regularly during incubation. Trypsin was inactivated in high-glucose DMEM. Samples were filtered through a 40 mm filter, followed by centrifugation for 10 min at 150 RCF. 10 000 cells in 0.04% BSA in HBSS with good viability (> 70%) per sample were targeted for analysis with Chromium X. Small intestines from wild type (WT) mice were incubated in 10 mM EDTA pH 8.0 for 10 min on ice. Cells were detached by scraping with a coverslip. After washing with PBS and centrifugation for 5 min at 150 RCF, cells were incubated in 4 mg/ml dispase II (Sigma, #04942078001), 1 mg/ml collagenase I (Worthington, #LS004196), 1 mg/ml collagenase H (Roche, #11074032001), and 1000 U/ml benzonase (ChemCruz, #sc-202391) in HBSS (Gibco, #14175-053) for 30 min at 32°C. This was followed by washing with cold DMEM, filtering once with a 100 nm filter and twice with a 40 nm filter, and centrifugation for 10 min at 150 RCF. 10 000 cells in 0.04% BSA in HBSS, with good viability per sample, were targeted for 10X chromium. The ScRNA-seq experiment was repeated 3 times, and the data were pooled from four mice. Library was performed according to the manufacturer`s instructions (single cell 3' protocol, 10x Genomics)