GSM5702474: TD2  [ST]; Homo sapiens; RNA-Seq
A human tumour dissociation kit enzyme solution (Miltenyi Biotec; 200 µl of H-enzyme, 100 µl of R-enzyme, 25 µl of A-enzyme, 4.7 ml of DMEM) was added for enzymatic digestion for 30 minutes at 37°C, and the sample was filtered through a Miltenyi 70-mm sieve. After centrifugation, the granular cells were suspended in erythrocyte lysis buffer. Finally, the cells were mixed with 1 ml of PBS, and the numbers of live cells and aggregated cells were measured with an automatic cell counter For droplet-based scRNA-seq, GemCodeGel beads, chips and library kits (10x Genomics) were used to process single cells according to the gel beads in emulsion (GEMs) protocol (Genergy Inc., Shanghai, China). Based on the manufacturer's instructions, the Chromium Single Cell 3' V2/V3 Kit was used to construct the scRNA-seq reagent library. Then, the cell suspension generated from each sample was reacted with certain reagents to generate single-cell GEMs in the Chromium Controller for sample and cell barcoding, with a target output of 8,000 to 12,000 cells per sample. The amplified cDNA and final library were evaluated with an Agilent Bioanalyzer using a high-sensitivity DNA kit (2100, Agilent Technologies). Then, the libraries were assembled and sorted on a NovaSeq 6000; approximately 400 M readings were obtained for each sample.