GSM5820668: N1; Homo sapiens; RNA-Seq
Samples from the tumor and adjacent non-malignant oral cancer tissue from the same resection specimen were isolated and saved in storage buffer. Each sample was subsequently minced on ice to less than 1 mm cubic pieces, followed by enzymatic digestion using type 2 collagenase (Sigma) with manual shaking every 5 min. Samples were then centrifuged at 300 relative centrifugal force (RCF) for 30 s at room temperature and removed the supernatant without disturbing the cell pellet. Next, 1× PBS (calcium and magnesium free) containing 0.04% BSA (400 µg/ml) was added and then centrifugation at 300 RCF for 5 min. The cell pellet was re-suspended in 1 ml red blood cell lysis buffer and incubated for 10 min at 4℃. After lysis of red blood cells, samples were re-suspended in 1 ml PBS containing 0.04% BSA. Next, samples were filtered over 40-µm cell strainers (VWR) to obtain single-cell suspension. After tumor dissociation, cell concentration and cell viability were determined by hemocytometer and trypan blue staining. RNA libraries were prepared for sequencing using standard Illumina protocols