title: Illumina HiSeq 2500 sequencing; Tissue inflammation reprograms lymphatic collectors to support entry and rapid migration of dendritic cells to draining lymph nodes
design: Tissue inflammation reprograms lymphatic collectors to support entry and rapid migration of dendritic cells to draining lymph nodes
library_construction_protocol: CHS-inflamed and uninflamed ears from individual animals were cut into small pieces and enzymatically digested in a 4 ml mixture of 10 mg/ml collagenase IV (Invitrogen), 5 mg/ml dispase II (Roche), 0.1 mg/ml DNase I (Roche) and 1 mM CaCl2 (Sigma Aldrich) in PBS at 37°C for 15 min in rotation. Suspensions were passaged through a 40 um cell strainer (BD Biosciences) and resulting single-cell suspensions were stained at 4°C with APC/Cy7 anti-mouse CD45 (BioLegend), PE anti-mouse CD31 (BioLegend), APC anti-mouse Podoplanin (BioLegend), AF488 anti-mouse Lyve1 (eBioscience) and 7-AAD (BioLegend). Different EC populations were isolated on a FACSAria Cell Sorter (70 um nozzle, BD Biosciences) and sorted into PicoPure RNA extraction buffer (ThermoFisher Scientific). CHS-induced ear skin inflammation. Briefly, 7-8-week-old C57BL/6 females were sensitized by topical application of 2% oxazolone (4-ethoxymethylene-2-phenyl-2-oxazoline-5-1, Sigma Aldrich) on shaved abdomen and paws; five days later 1% oxazolone was applied on ears and 24h later animals were sacrificed and ears were harvested. RNA was isolated and genomic DNA was eliminated using the PicoPure RNA isolation kit (ThermoFisher Scientific). RNA quality and concentration were analyzed with a Bioanalyzer (Agilent Technologies) and samples of the same animal with RIN above 7 and minimum concentration of 200 ng/ml were further processed for RNA sequencing. cDNA libraries were generated using Ovation Single Cell RNA‐Seq System (NuGen) and the quality and concentration of resulting libraries were assessed with a Bioanalyser.