GSM5394134: post_m02: Mouse 2 posterior jejunum; Mus musculus; OTHER
The mouse small intestines were dissected in 12 equal pieces. The 3rd (anterior jejunum) and 7th (posterior jejunum) parts were used for single cell RNA-sequencing. The small intestines were cut in pieces of 1 cm2, placed into 50 ml conical tubes and washed 3 times for 20 min with 25 ml of PBS/ 5 mM EDTA on a rocking plate at room temperature. Villi were mechanically removed by gentle shaking 3 times and trituration using a 25 ml pipette. Crypts were separated from the intestine by vigorous shaking 2 times. Both villi and crypts were collected by centrifugation at 200g for 5 min, washed twice with PBS. Cells from the villi and crypts were mixed at 1:1 ratio. To isolate epithelial cells, single cell suspensions were stained with APC–conjugated anti-EpCAM 1:1000 (eBioscience) antibody for 30 min at room temperature. Fluorescence-activated cell sorting was performed using BD FACS Aria III SORP cell sorter (100 μm nozzle). Living cells were gated by DAPI dye exclusion. Single EpCAM+ cells were sorted in 386 well plates. Single cell libraries (pooled) were constructed using the MARS-Seq protocol as described by Jaitin DA, et al. Science 343, 776–9 (2014).