title: Single cell RNAseq of fibroblastic reticular cells from mesenteric and inguinal murine lymph nodes at different developmental stages
design: Single cell RNAseq of fibroblastic reticular cells from mesenteric and inguinal murine lymph nodes at different developmental stages
library_construction_protocol: 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 Chemistry).
identifiers.namespace: European Bioinformatics Institute
identifiers.id: E-MTAB-13891_2:Sample 1_p