Females 10–11-month-old mice were i.p. administered a lethal dose of ketamine (100 mg/mL)/xylazine (10 mg/mL), intracardially injected with 10 µl of heparin sodium and transcardially perfused with ice-cold 1x PBS. The dCLNs, sCLNs and iLNs were harvested and placed into a 0.4 ml enzymatic mix [100 µg/ml Liberase TL (Roche) and 20 µg/ml DNase I (Roche) in sterile freshly opened IMDM (Sigma)] in a 24-well plate on ice. The tissues were mechanically dissociated using sterile 1 ml syringe plungers on ice and incubated at 37°C for 10 minutes with frequent agitation. The enzymatic reaction was stopped by placing the plate on ice and adding ice-cold RPMI (Biological Industries) supplemented with 10% FBS (Gibco) and 2 mM EDTA (Biological Industries). Samples were then filtered through a 100 µm filter and centrifuged at 450 x g for 6 minutes. The supernatant was aspirated and BioLegend cell staining buffer was used to wash and resuspend the cells. Cell count was evaluated using an automated cell counter (TC20, Bio-Rad). From each sample, 2*106 cells were transferred into new tubes and washed with 1 ml of BioLegend cell staining buffer. After aspiration of the supernatant, the residual contents of each sample were resuspended and incubated with 0.5 µl TruStain FcX PLUS (anti-mouse CD16/32, BioLegend) blocking reagent at 4°C for 10 minutes. Cell hashing was performed by incubating at 4°C for 30 minutes 0.5 µg of anti-mouse Totalseq-C 001, 002, 003 or 004 hashtags (oligo-tagged antibodies from BioLegend). Next, three washes with 3.5 ml of BioLegend cell staining buffer were performed. The cells were then resuspended with 1x PBS supplemented with 1% bovine serum albumin (BSA, Sigma), and cell viability was evaluated using Trypan blue and an automated cell counter (TC20, Bio-Rad). Only samples with cellular viability higher than 87% were used. The single cell suspension from the different tubes were finally combined into composite samples containing an equal number of cells from each original sample. Up to 17,500 cells from each composite sample (final concentration ~1000 cells/μl) were loaded onto a 10x Genomics Chromium platform for gel beads-in-emulsion (GEM) partitioning and cDNA generation. Gene Expression (GEX), V(D)J (TCR and BCR) and Cell Surface Protein library from the same samples were then constructed using the Chromium Next GEM Single Cell 5' Reagent Kit v2 (Dual Index) with Feature Barcode technology for Cell Surface Protein and Immune Receptor Mapping. Libraries were sequenced on the NovaSeq 6000 (Illumina), achieving library depths of ~22,200-36,800 mean reads per cell for GEX, ~13,100-26,400 reads per cell for TCR, ~10,900-26,500 mean reads per cell for BCR, and ~4,400-8,400 mean reads per cell for feature barcodes (cell hashing).