title: GSM5484299: brachial LN E17.5; Mus musculus; RNA-Seq
library_construction_protocol: For the scRNAseq performed on E17.5 LNs:  Brachial, inguinal and mesenteric lymph nodes were harvested from E17.5 C57BL6/N wild-type embryos into RPMI (Fisher Scientific) with 2% FBS (EuroClone), 20mM HEPES (Fisher Scientific). Tissue were transferred to room temperature digestion buffer (0,8mg/ml Collagenase/Dispase (Roche) + 100ug/ml DNAse I (Roche) in RPMI with 2% FBS and 20mM Hepes) and incubated at 37°C for 30 minutes at 900rpm in thermomixer. Every 10 minutes, tissues were placed on ice, large pieces were settled and supernatant were transferred into a new tube with MACS buffer; next larger fragments were pipetted with fresh digestion mix and placed into thermomixer for the remaining time. The digested cell suspension was centrifugated, then cells were counted and stained with anti-CD45, anti-VCAM1 and anti-ICAM1 for sorting. Sorted cells CD45-/VCAMhi/ICAMhi cells were spun down and immediately run on the 10X Chromium (10X Genomics) and then processed through library preparation following the recommended protocol for the Chromium Single Cell 3' Reagent Kit (v2 Chemistry, 10X Genomics). For the scRNAseq performed on adult LNs: Brachial, inguinal and mesenteric lymph nodes were harvested from C57BL6/N wild-type mice at 2 months of age into RPMI (Fisher Scientific) with 2% FBS (EuroClone), 20mM HEPES (Fisher Scientific). Tissue were transferred to room temperature digestion buffer (0,2mg/ml Collagenase (Roche) + 0,45mg/ml Liberase (Roche) + 100ug/ml DNAseI (Roche) in RPMI with 2% FBS and 20mM Hepes) and incubated at 37°C for 30 minutes at 900rpm in thermomixer. Every 10 minutes, tissues were placed on ice, large pieces were settled and supernatant were transferred into a new tube with MACS buffer; next larger fragments were pipetted with fresh digestion mix and placed into thermomixer for the remaining time. The digested cell suspension was centrifugated, cells were counted and resuspended in MACS buffer with anti-CD45-APC antibody + anti-APC MACS microbeads (Miltenyi) to deplete CD45+ cells. Eluted CD45- cells were spun down, counted and stained for anti-CD45, anti-CD31 and anti-GP38 for sorting. Sorted stromal cells (CD45-/GP38+) and endothelial cells (CD45-/CD31+ and CD45-/GP38+/CD31+) were spun down and immediately run on the 10X Chromium (10X Genomics) and then processed through library preparation following the recommended protocol for the Chromium Single Cell 3' Reagent Kit (v2 Chemistry, 10X Genomics). Chromium Single Cell 3' Reagent Kit (v2 Chemistry, 10X Genomics)