Tumor Dissociation and Single-cell Analysis

Following resection, a representative tumor fragment was isolated and transferred rapidly to the laboratory for study as previously described [20 (link)]. Briefly, tissue was initially cut into segments and subjected to digestion by collagenase type I/II (Thermo Fisher Scientific, USA) and DNAse I (Sigma, USA). The digested pieces were triturated with a 1 ml syringe plunger and passed through a 70 μm cell strainer (Coring, USA). After resuspending in red blood cell lysis buffer (Solarbio, China), live cells were enriched using a Dead Cell Removal kit (Miltenyi Biotec, Germany) as per manufacturer’s instructions. Enriched live cells were washed and counted using a hemocytometer with trypan blue. Cells were then resuspended in PBS containing 0.04% BSA at a concentration of 1 × 10⁶ cells/ml with a viability of > 80% as determined with the Countess. Overall, the entire dissociation procedure took about 2 h from obtaining sample to generating single-cell suspension. The single-cell suspension was then run on the Chromium 10X device (10 × Genomics, USA).
Single-cell library preparation was carried out using Chromium Single cell 3’ Reagent v2 Kits (10 × Genomics, USA) according to the manufacturer’s protocol. Then the library was sequenced on the HiSeq X Ten instruments (Illumina, USA) and 150 bp paired-end reads were generated.