GSM5418894: LApcL; Mus musculus; RNA-Seq
Small intestine of WT, LApc and LApcL mice was removed and washed with cold PBS. Intestines were cut into 3-4 pieces and incubated in 10 mM EDTA in orbital shaker +4°C for 30 min. Tissues were washed with PBS and cells from luminal surface were scraped off with a glass slide. Cells were then centrifuged 300 rpm +4°C followed by incubation with enzymes (4 mg/ml Dispase II, 1 mg/ml Collagenase I, 1 mg/ml Collagenase H and 1 U/ml Dnase I) for 7 min at 32 °C. Cells were washed with DMEM with 2 % FBS and centrifuged at 300 rpm +4°C for 2 min after which floating cells were collected and filtered through a 40 μm mesh. Remaining cells were centrifuged at 300 rpm +4°C for 5 min, and incubated in trypsin-EDTA with 1 U/ml DNase I for 5 min at 32°C. After a wash with DMEM with 2 % FBS, the cells were centrifuged at 300 rpm +4°C and filtered through a 40 μm mesh. Epithelial cells of WT, LApc and LApcL mice were sorted on BD Influx (BD Biosciences). The epithelial cells were surface-stained with Anti-Mo CD326 (EpCAM) PE (eBioscience, Invitrogen #12-5791-82). Sorted cells were resuspended into HBSS with 0.04 % BSA. Sample libraries were sequenced using the Illumina NovaSeq 6000 system (50,000 reads per sample).