GSM6267872: Pheripheral blood cells, patient with prostate cancer, CITE-RNA seq; Homo sapiens; RNA-Seq
Peripheral white blood cells (pWBCs) were gathered as below. Blood samples were collected in heparin blood collection tubes, and red blodd cells were removed usinig Hetasep (STEMCELL Technologies).pWBCs were enriched by density gradient centrifugation at 90 x g for 4 minutes at room temperature. pWBCs were labeled by TotalSeq-B Human TBNK Cocktail (BioLegend).Library was performed according to the manufacter's instructions (Chromium Single Cell 3' Reagent Kits v3.1, 10x Genomics) for scRNA-seq and CITE-seq. Briefly, pWBC were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRI select reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.