GSM6246188: control-PBMC, replicate 2, scRNAseq; Homo sapiens; RNA-Seq
Fresh tumor lesions were stored in RPMI medium and processed on ice after the surgery within 30 mins. The specimens were washed with Hanks Balanced Salt Solution (HBSS) three times and minced into 1–2 mm pieces. Then, added mixed Enzyme according to Tumor Dissociation Kit protocol (Miltenyi Biotec:130-095-929) and performed the association process in gentleMACS™ Dissociator (Miltenyi Biotec:130-093-235). After digestion, the samples were filtered through 40-µm sterile strainers and centrifuged at 800 × g for 5min. Subsequently, the supernatants were discarded, and the cell pellets were suspended in 1 mL phosphate buffered saline (PBS; HyClone, United States). To remove red blood cells, 2 mL of red blood cell lysis buffer (eBioscience™00-4333-57) was added, and cells were incubated at 25 °C for 10 min. The solution was then centrifuged at 500 × g for 5 min and resuspended in PBS. The samples were stained with trypan blue (Sigma, United States) and the cellular viability was evaluated under the phase contrast light microscope (Nikon, Japan). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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 SPRIselect 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.