GSM7979096: Ileum_CD; Mus musculus; RNA-Seq
The small intestine was removed from mice and divided into three parts representing the duodenum, jejunum and ileum from proximal to distal. Intestines were washed in ice-cold PBS by vortexing. Then, the samples were incubated for 30 min at 37 °C with horizontal rotation in 20 ml predigestion solution (1× HBSS without magnesium/calcium, 10 mM HEPES, 5 mM EDTA, 1 mM DTT and 2% FCS). IECs were detached by intensive vortexing and harvested on ice using a 100μm cell strainer (Greiner). To increase the IEC yield, samples were vortexed a second time in 10 ml predigestion solution, and cell suspensions were pooled. Afterwards, IECs were collected by centrifugation (10 min at 50 g and 4 °C) and resuspended in 1X HBSS/2%FCS solution. Isolated cells from three 12-week-old male mice were pooled in order to minimize artefacts originating from individual mice. IECs and intraepithelial immune cells were resuspended in 2% FCS in PBS and counterstained using propidium iodide (PI). Live cell sorting was performed with a BD FACSAria Fusion and BD FACSAria IIIu at 4°C using a 100 μm nozzle and sheath pressure set at 20 psi. Live single cells were gated based on PI fluorescence. Sorted, PI negative cells were collected in 1xPBS buffer containing 0.04% BSA for 10X genomics single cell sequencing. Isolated cells were processed using a 10X Genomics Chromium Controller. cDNA was prepared using the Chromium single-cell 3′ reagent kit v3 according to the manufacturer's protocol (10X Genomics).