GSM5804902: CD45-CD31- cells - resting Lymph Node; Homo sapiens; RNA-Seq
Fresh lymph nodes and tonsils were cut into small pieces and treated with an enzymatic cocktail containing collagenase D (1 mg/mL; Sigma Aldrich), DNase I (0.1 mM; Roche) and Dispase I (2 U/mL; Roche) in serum-free RPMI for 40 minutes at 37°C. The resulting cell suspension was passed through a 100 µm-mesh filter and the cells were washed in phosphate-buffered saline (PBS) containing 6 mM EDTA. Red blood cells were removed by magnetic isolation in EasySepTM RBC Depletion Reagent (Stem Cell Technology) according to the manufacturer's instructions. The remaining cells were then washed in 10% fetal calf serum (FCS) and 2 mM EDTA in PBS. Immune cells were removed by magnetic isolation with a human CD45 microbeads kit (Miltenyi) use according to the manufacturer's instructions. The resulting CD45- cell-enriched suspension was washed with PBS and stained directly. The CD45- cell-enriched suspension was stained by incubation with a Live/Dead fixable yellow fluorescence kit (Invitrogen, 1:200) in PBS for 20 minutes at room temperature before surface staining. Cells were then incubated for 40 minutes at 4°C in PBS containing 1% FCS and 2 mM EDTA plus the following antibodies: APC-Cy7 anti-CD45 (BD Biosciences), FITC anti-CD31 (BD Biosciences), biotinylated anti-FDC (Thermo Fisher Scientific) and PE anti-PDPN (Thermo Fisher Scientific). The biotinylated antibody was detected with streptavidin conjugated BV421 (Biolegend). Viable FRCs and epithelial cells were sorted by flow cytometry (ARIA III, BD) on the basis of their CD45-CD31- phenotype. This CD45-CD31- cell fraction was enriched in FDCs and non-FDC-FRCs by sorting PDPN+CNA.42+ cells and PDPN+CNA.42- cells, respectively. PDPN-CNA.42+ and PDPN-CNA.42- cells were also sorted to obtain tonsillar epithelial cells and to identify other CD31-PDPN-FRCs subsets. Sorted CD45-CD31- cells were analyzed with the 10X chromium system (10X Genomics). Single-cell libraries were generated according to the manufacturer's instructions (chromium 10X Next GEM Single-cell 3' Kit (V.3.3)). Next-generation sequencing was performed on an Illumina NovaSeq 6000. ScRNAseq data processing and FASTQ file analysis were performed at the NGS platform of the Curie Institute, Paris (Tonsil 1) and the sequencing platform of the Paris Brain Institute (Tonsil 2-4, LNs) with the Cell Ranger pipeline (10X Genomics, version 3).