GSM4849547: C3KOC; Mus musculus; RNA-Seq
Following dissection, colon tissue was placed in ice-cold PBS immediately and maintained on ice for further processing. Mesenteric material, fat, and Peyer's patches were removed. Colon sections were cut open, feces removed, and colon tissue was cut into 1-2 cm pieces. Colon tissue pieces were placed in 8 mL ice-cold PBS and shaken vigorously by hand at room temperature for 2 min. Tissue pieces were transferred to 20 mL PBS containing 1 mM DTT (Sigma), 30 mM EDTA (Sigma), and 10 mM HEPES (Sigma) at room temperature. The samples were placed in a bacterial shaker incubator horizontally, rotated at 37 ℃ for 10 min at 200 rpm, and then shaken by hand at room temperature for 1 min40. Tissue pieces were transferred to new tubes containing a fresh solution of 20 mL PBS, 30 mM EDTA, and 10 mM HEPES, and were shaken for a second 10 minutes as indicated. The tissue pieces were rinsed gently with RPMI medium containing 2% heat-inactivated FBS (RPMI/2% FBS) at room temperature. Tissues pieces were transferred to tubes containing 5 mL of RPMI medium, 10% FBS, 200 U/ml collagenase VIII (Sigma), and 150 μg/ml DNase I (Sigma). Tissues were digested by shaking in a bacterial shaker incubator at 100 rpm at 37 ℃ for 1 h, and then shaken by hand at room temperature for 1 min. The digested tissues were placed on a 100 μm cell strainer; single cells in the flow-through were collected. Cells were centrifuged at 600 g for 5 min at 4℃ and resuspended in 4 mL 40% Percoll (Sigma) in RPMI/2% FBS. The Percoll-cell solution was layered on top of 4 mL 80% Percoll in RPMI/2% FBS, and centrifuged at 1300 g at room temperature for 20 min; spins were concluded without using the centrifuge brake. The cells at the interface of the 40% and 80% Percoll solutions were collected; cells were washed once with ice-cold PBS/2%FBS prior to antibody incubations.Colonic LP immune cells were isolated and sorted for live CD45+ cells by fluorescence-activated cell sorting (FACS). https://assets.ctfassets.net/an68im79xiti/4tjk4KvXzTWgTs8f3tvUjq/2259891d68c53693e753e1b45e42de2d/CG000183_ChromiumSingleCell3__v3_UG_Rev_C.pdf