GSM8893198: IEL_mast_cell, replicate 1; Mus musculus; RNA-Seq
Single-cell suspensions of small intestinal epithelium and lamina propria were prepared as described . Briefly, the small intestine was isolated and opened longitudinally. Its contents were then rinsed in PBS following removal of Peyer's patches. The tissue was then cut into 2-3 cm segments and incubated in RPMI media (ThermoFisher) containing 5 mM EDTA, 0.145 mg/mL DTT, and 3% FBS at 37°C with 5% CO2 for 20 min with agitation. Pieces of intestine were washed in RPMI containing 2 mM EDTA to separate the epithelial fraction. Lamina propria digestion was performed using 0.1 mg/mL Liberase TL (Roche #540102001) and 0.5 mg/mL DNAse (DN25, Sigma-Aldrich) in RPMI for 30 min at 37°C with 5% CO2. Digested tissue was sequentially strained through 70 mM and 40 mM strainers and washed in RPMI containing 3% FBS. The epithelial fraction was also subjected to a 30% Percoll density gradient by centrifugation (GE17-0891-01, Sigma Aldrich) in preparation for sorting.  Cells were then stained and sorted via FACS Aria into ice cold 10% FBS in dPBS w/o Ca, Mg, or EDTA. .  Stain:  Fc block, EMA (live/dead), FceRI-PE, CD11b-APC, CD19-APC/efluor780, CD45-efluor450, CD3-Alexa700, CD117-PE/Cy7.  MCs were gated as live, CD45+ CD19- CD11b- CD3- CD117+ FceRI+ GEM-X Single Cell 3' v4 assay (10X Genomics) according to the manufacturer's protocol