GSM8172950: EU_SUP,scRNAseq; Homo sapiens; RNA-Seq
Tissues were isolated by mincing the freshly obtained surgical specimens into 1-mm cubic pieces followed by enzymatic digestion using 2mg/ml collagenase II, 2mg/ml collagenase IV, and 0.1mg/ml DNase I, and were incubated on a rocker for 30 min at 37 °C. The digested tissues were then passed through a 40 µm cell strainer and washed twice with PBS prior to surface staining. Determine cell concentration using AO/PI fluorescent staining dyes.The isolated cells are then loaded directly into compatible 10x Genomics Single Cell assays. Following cell isolation, samples were sequenced by 10x Chromium single-cell platform (10x Genomics, Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 chemistry, Rev D). In detail, Gel Beads-in-emulsion (GEMs) generation and barcoding was first performed. Then, post GEM-RT cleanup and cDNA amplification were conducted. Finally, the 3′ gene-expression library construction was performed following the manufacturer's instructions. The library is generated and sequenced on NovaSeq6000 (Illumina).