GSM7789318: RelBdDC mLN steady state; Mus musculus; RNA-Seq
the small intestine was removed, placed in ice-cold DPBS  and Peyer's patches were removed. Intestines were cut open longitudinally, washed with ice-cold PBS, and cut into 0.5 - 1 cm pieces. For mucus removal, small intestine pieces were incubated in RPMI 1640 (Gibco) containing 25 mM HEPES (Gibco), 5 mM EDTA (Invitrogen), 3% FCS (Sigma Aldrich), and 0.145 mg/ml DTT (Sigma) for 20 min at 37°C, 80 rpm. Thereafter, tissues were washed three times with RPMI1640 containing 25 mM HEPES and 2 mM EDTA and minced with sharp scissors into 1-2 mm pieces. To generate of single cell suspensions, minced pieces were digested for 30 min by shaking at 100 rpm at 37°C in RPMI 1640 containing 25 mM HEPES, 200 iU/ml Collagenase IV (Worthington), and 0.2 mg/ml DNaseI (Sigma). Tissue pieces were then pipetted up and down several times and cell suspensions were collected. One more cycle of digestion with fresh medium was performed and cell suspensions were filtered through a 100 µm cell strainer (Corning). Cell suspension was centrifuged at 450 g, for 5 min at 4°C, cell pellets were resuspended in 40% Percoll (Cytiva) and layered onto an 80% Percoll solution. The Percoll gradient was centrifuged at 1600 g at room temperature for 15 min. The interlayer containing lamina propria cells were used for FACS. Cells were stained with antibodies against CD45, CD64, B220, CD11c and MHCII and before sorting 7AAD was added to label dead cells. They were then sorted for CD45+7AAD-, CD64-, B220-, CD11c+ MHCII+ using the FACS Aria Fusion and collected in PBS with 0.04% BSA. For mesenteric lymph nodes, organ was collected and cut into 1-2 mm pieces. Tissue pieces were digested by incubation in RPMI 1640 containing 25 mM HEPES, 200 iU/ml Collagenase IV (Worthington), and 0.2 mg/ml DNAseI (Sigma) for 30 min at 37°C, shaking at 100 rpm. After washing, cell suspension was stained in a similar manner as SI-LP cells. Library was prepared according to manufacturers instructions (Chromium Next GEM Single Cell 3' Kit v3.1; 10X Genomics)