GSM5196867: Immue cells from small intestine, cecum and colon of mouse young-5 and mouse old-5; Mus musculus; RNA-Seq
Immune cells were isolated from lamina propria: tissue (cleared of crypts) was chopped finely with a scalpel and then incubated in 3ml of 1mg/ml collagenase-D (Merck, Cat. 11088866001) and 1mg/ml DNaseI in RPMI medium (ThermoFisherScientific, Cat. 21875091) in 6 well plates in an incubator shaker (80rpm) (VWR, Cat. 444-0732) for 20min at 37°C. Tissue pipetted up and down several times with a cut p1000 tip. The supernatant was passed through a 100μm strainer into 5% FBS in RPMI. Leftover tissue incubated again twice in 3ml collagenase-D/DNaseI RPMI solution in an incubator shaker (80rpm) for 30min at 37°C, collecting filtered supernatant. After the third incubation, the remaining tissue was smashed with a syringe plunger on a Falcon 40μm cell strainer (Corning, Cat. 352340) and washed with 5% FBS/RPMI to collect the maximum number of cells. The supernatant was centrifuged at 300g, 4°C for 5min. The pellet was re-suspended in 40% percoll (Sigma-Aldrich, Cat. P1644-1L) in RPMI. The cell suspension was carefully pipetted over 80% percoll in a falcon tube in order to create a gradient. The falcon tubes were centrifuged at 1600g, RT for 20min (centrifuge break disabled). The immune cells formed a white ring on the border of the two percoll concentrations: they were collected carefully and washed with FSM (2% FBS in PBS). The suspension was centrifuged at 450g, 4°C for 5min. Resulting pellet was resuspended in FSM and transferred for staining. The 10X Single Cell 3' v2 or v3.1 reagent kit (10X Genomics) was used for cell partitioning, cDNA amplification and library construction.