GSM5997536: Ahrr wt2; Mus musculus; RNA-Seq
IEL and LPLs were isolated from small intestinal epithelium and lamina propria respectively. Small intestines were flushed with HBSS to remove the fecal content. Peyer's patches were removed followed by longitudinal opening and cut into 1cm pieces. Then, the pieces were washed by gently agitating for 20 minutes followed by vertexing. The flow through was collected and the cycle repeated once again. The flow through was subjected to DTT treatment and Percoll 40-70% gradient separation for isolation of IEL. For LPL isolation, the pieces were rinsed properly with HBSS and then proceeded to digestion with complete RPMI supplemented with collagenase-IV. The undigested tissue chunks were removed by passing through 100-micron mess and then the digests were subjected to 40%-70% Percoll gradient centrifugation for isolation of LPL. WT and Ahrr–/– IEL (CD45+, CD3+, CD19–) were sorted using BD FACS ARIA-II. Sorted cells were sequenced using the10X Genomics Single Cell 3' v3 platform.