GSM7265930: Donor 1 Hashtag scRNAseq; Homo sapiens; RNA-Seq
PBMC isolation was performed by density centrifugation (500 g, 30 min, 21 °C) using Lymphocyte Separation Media (catalog no. LSM-A, Capricorn, Ebsdorfergrund, Germany). After collection of PBMCs at the interphase of the layers and washes using Hank´s Balanced Salt (catalog no. H6648, Sigma Aldrich, St. Louis, MO, USA), remaining erythrocytes were lysed with ASK Lysing Buffer (catalog no. BP10-548E, Lonza, Basel, Switzerland) for 3 min. Cell counts were determined using the Trypan Blue (catalog no. T8154, Sigma Aldrich) exclusion method on a TC20 Automated Cell Counter (Bio-Rad, Hercules, CA, USA) and aliquots of different proportions were prepared. PBMCs were frozen in heat inactivated fetal bovine serum (FBS, catalog. no S0615, Sigma Aldrich) + 10% dimethyl sulfoxide (catalog no. D85897, Sigma Aldrich) at -80 °C and transferred into liquid nitrogen storage the next day until further processing. For fluorescence-activated cell sorting of pDCs, PBMCs were stained with surface antibodies and a dead stain (NEAR-IR) for 20 minutes at room temperature in the dark. Cells were loaded onto the 10X Chromium Controller (10X Genomics, Pleasanton, CA, USA). Single-cell libraries were generated using the Chromium Next GEM Single Cell 5' Kit v2 (10X Genomics) according to the manufacturer's instructions. Libraries were sequenced on an Illumina NovaSeq 6000 system (S4 flow cell) with 150 base pairs and paired-end configurations.