GSM8391115: P03_PBMC,Pre, scRNAseq; Homo sapiens; RNA-Seq
Freshly biopsied tumor tissues were flushed with 1×HBSS (Mg2+ Ca2+ free supplemented with 0.04% BSA) to remove any residual blood. Tumor-infiltrating cells isolation was performed by adding digestion buffer into tissues (1 mg/ml Collagenase I, 1mg /ml neutral protease, 0.2 mg/ml DNase I, and 0.01 % Hyaluronidase) in a shaking incubator at 37°C for 30 - 100 minutes. The isolated cells were resuspended with 1 × red blood cell removal solution and placed on ice for five minutes. Cells were washed and re-suspended in PBS then stained with a Viability Dye. The cell suspension was diluted with 1 × HBSS till a concentration of 700 - 1200 cells/μL was reached. Library was constructed according to the manufacter's instructions (single-cell 3' v2 protocol, 10×Genomics). Briefly, tumor-infiltrating cells were resuspended in the master mix; they were then loaded with partioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate in every single GEM was retrotranscripted to DNA, which contained lumina R1 primer sequence, Unique Molecular Identifier (UMI), and the 10×barcode. The pooled barcoded cDNA was then cleaned up with Silane Dynabeads and amplified by PCR; the appropriately sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction, lumina R2 primer sequence, paired-end constructs with P5 +P7 sequences and a sample index were added.