GSM8841600: COVAX_015R_GEX; Homo sapiens; RNA-Seq
FNB lymph node samples (n=12, which includes samples for both LNs for each of 6 participants) were washed with DPBS by centrifugation at 550 x g for 5 mins. The supernatant was discarded, and the cell pellet was resuspended in 100µl PBS. To confirm the presence of lymphocytes, cells were stained with CD45-AF700 (clone HI100) (BD Pharmingen) for 15 mins at RT in the dark, RBCs were lysed with 2.4mL RBC Lysis Solution (16.52g NH4Cl + 2g KHCO3 + 0.74g disodium EDTA in 2L dH2O, pH7.2-7.4) for 10 mins at RT in the dark, with intermittent agitation. Samples were then centrifuged at 550 x g for 5 mins. The supernatant was discarded, and the cells were resuspended in 100µl Iscove's Modified Dulbecco's Medium (IMDM) + 2% FCS. Prior to sorting, the cells were stained with DAPI (0.1μg/ml) (Sigma Aldrich) for 15mins in the dark and live-sorted on a FACSAriaIII cell sorter (BD Biosciences). Single cell libraries were generated using the 10X Genomics 5'v2 Gene Expression and Immune Profiling at the Garvan Cellular Genomics Platform, according to manufacturer's instructions. cDNA and BCR libraries were then sequenced using the Illumina NovaSeq S4 Flow Cell according to manufacturer's instructions at Read 1 = 28, i7 index = 10 and Read 2 = 90 cycles.