Single cell RNAseq of fibroblastic reticular cells and vascular smooth muscle cells from mesenteric and peripheral murine lymph nodes at different developmental stages
Single cell RNAseq of fibroblastic reticular cells and vascular smooth muscle cells from mesenteric and peripheral murine lymph nodes at different developmental stages
For stromal cell isolation from peripheral (inguinal, brachial and axillary) and mesenteric lymph nodes, tissues were disrupted into small pieces using small needles and collected in RPMI 1640 medium containing 2% FCS, 20 mM HEPES pH 7.2 (Lonza), 0.16 mg/ml collagenase P, Dispase 30 μg/ml (Roche) and 25 μg/ml DNase I. The dissociated tissue was incubated for 45 min at 37 °C, during the incubation time the tissue was resuspended and supernatant was collected every 15 min.  Enrichment of stromal cells was performed by incubating the cell suspension with MACS anti-CD45 and anti-TER119 microbeads (Miltenyi Biotec) and passing it through MACS LS columns (Miltenyi Biotec). Single-cell suspensions were stained for further cell sorting. Stromal cells were sorted for CD31-EYFP+ cells and were filled up with CD31-EYFP- cells. Cell sorting was performed using a BD FACSMelody Cell Sorter and the FACSChorus (v.1.3) software (BD Biosciences). cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3.1 Chemistry). 