GSM9488533: scRNA, PBMC, protein vaccinated_1; Homo sapiens; RNA-Seq
Peripheral blood was obtained from vaccine recipients and mononuclear cells were isolated from 16 ml of peripheral blood using Vacutainer® CPT™ Mononuclear Cell Preparation Tube (Becton, Dickinson and Company, NJ, USA). After collecting 4 ml of plasma, the white opaque layer containing PBMCs was isolated and washed with RPMI supplemented with 10% FBS twice. The isolated PBMCs were frozen in CELLBANKER®1 Cell Freezing Media (Zenogen Pharma, Fukushima, Japan) for liquid nitrogen storage. After, PBMC was thawed and suspended in a 2 ml tube, then centrifuged at 4°C for 5 min at 500 x g in a swinging bucket centrifuge. The pellet was resuspended in 100 ul of Lysis Buffer and incubated for 3 min on ice. After incubation, 1 ml chilled Wash Buffer was added to lysed cells and mixed with pipetting. The sample was centrifuged at 4°C for 5 min at 500 x g. The supernatant was removed without disrupting the nuclei pellet, and this washing step was repeated two more times for a total of 3 times. For vaccinated samples, nuclei from four donors were multiplexed per GEM, with 12,500 nuclei loaded per donor. For non-vaccinated samples, 15,000 nuclei were loaded per GEM without multiplexing. All samples were processed on the Chromium Next GEM Chip J (PN-1000230) according to the manufacturer's recommendations. All single-cell libraries were produced using the 10x Chromium Single Cell ATAC + Gene Expression workflow following the manufacturer's instructions (PN-1000283). Final libraries were sequenced using Illumina NovaSeq 6000(Illumina, Inc., San Diego, CA, USA), for both GEX and ATAC sequencing.