Single cell RNAseq of immune and stromal cells from clinically inflammed and non inflammed human lymph nodes
After surgical excision torn tissues were collected sterile phosphate buffered saline on ice and processed within one hour of resection. For enzymatic dissociation of human lymph nodes, torn tissues were collected in RPMI 1640 medium containing 2% FCS, 20 mM HEPES pH 7.2 (Lonza), 2.4 mg/ml Dispase (Roche),  0.375 mg/ml Collagenase P (Roche) and 25 mg/ml DNaseI (Applichem). To enrich for stromal cells, hematopoietic cells and erythrocytes were depleted by incubating the cell suspension with MACS anti-CD45 and anti-CD235a microbeads (Miltenyi Biotec) and passing it through a MACS LS column (Miltenyi Biotec). The unbound single cell suspension was stained for further cell sorting. For isolation of hematopoietic cells, tissues were gently smashed across a 26-gauge wire mesh and washed with medium (RMPI-1640 containing 2% FCS (Sigma), 1% Penicillin-Streptomycin (Corning) and 20mM HEPES (Sigma)) until further staining for cell sorting. Hematopoietic cells were stained with fluorochrome-conjugated antibodies against human CD45, CD3, CD19 and CD14 and stromal cells with fluorochrome-conjugated antibodies against human CD45 and CD235a. Live/dead cell discrimination was performed by adding 7-amino-actinomycin D (7AAD; Calbiochem) prior to acquisition. CD45– CD235a– stromal cells, CD45+ CD3+ T cells and CD45+ CD19+ B cells were sorted with a BD FACS Melody cell sorter (BD Biosciences) and run on the 10x Chromium analyzer (10X Genomics). cDNA library generation was performed following the established commercial protocol for Chromium Single Cell 3' Reagent Kit (v3 Chemistry).