GSM7230522
Blood samples were diluted 1:1 with phosphate-buffered saline (PBS, Gibco) and layered onto the Ficoll Paque Plus (GE-healthcare) and centrifuged at 400g for 30 min, the buffy layer containing the peripheral blood mononuclear cells (PBMCs) were collected in 1.5mL centrifuge tubes. Cells were first isolated then washed twice in PBS by centrifugation at 10,000g for 4 min. Cell viability was determined using Trypan Blue and found to be greater than 9 Single-cell suspension samples were loaded on the Chromium platform (10X Genomics) for cell barcoding and library preparation was done using the Chromium Single Cell 5' Reagent kit (10X Genomics) using the manufacturer's instructions. Briefly, single-cell gel beads in emulsion (GEMs) generated from the Chromium controller were reverse transcribed to produce 10X barcoded cDNA. cDNA was then amplified, and the library generated. Sequencing was performed on the Illumina NovaSeq6000 platform according to Illumina standard conditions with 10X Genomics recommended read parameters.