GSM7978680: HC, replicate 1, scRNAseq; Homo sapiens; RNA-Seq
Freshly obtained resected tissues were rinsed with Hanks' Balanced Salt Solution (HBSS) after the operation, subsequently shredded on ice to smaller pieces with collagenase I/IV in HBSS, and incubated for 30 min at 37 °C with manual shaking every 10 min. The digested tissues were then passed through a 70-μm nylon mesh filter, and the cell suspension were centrifuged at 400 g for 5 min at 4 °C. After removing the supernatant, the pelted cells were suspended in red blood lysis buffer, and next resuspended in PBS after being washed with HBSS. The cDNA library was constructed within 24 h. We mixed a single-cell suspension with 0.4% trypan blue dye at a ratio of 9:1. Cells were counted using a Countstar Rigel S2. The proportion of living cells was calculated and should exceed 90% for quality control. Libraries were prepared according to the manufacturer's protocol (10x Genomics, CG000086_RevM). After quality checks, single-cell RNA-seq libraries were pooled and sequenced on a NovaSeq 6000 instrument.