Macs tumour dissociation kit

Single‐cell suspensions were obtained as previously described.⁴³ Briefly, fresh tumour samples were cut into pieces and digested with a MACS tumour dissociation kit (Miltenyi Biotec). Single‐cell suspensions were collected, and a Rigel S3 fluorescence cell analyser (Countstar) was used to assess cell viability and concentration. The cell suspension was loaded onto the Chromium single‐cell controller (10x Genomics). The scRNA‐seq libraries were constructed using the Single Cell 3′ Library and Gel Bead Kit v3.1 and sequenced using the Illumina NovaSeq 6000 sequencer (performed by CapitalBio Technology). scRNA‐seq data were aligned and quantified using the CellRanger toolkit v.3.1. Major cell types in the tumour tissue were clustered using Seurat (v.3.2.3). Differentially expressed genes were identified using FindMarkers. Enrichment analysis was performed using KOBAS software with Benjamini‒Hochberg multiple testing adjustment using the top 20 marker genes of the clusters. The outcomes were graphically represented utilizing the R software package.

The cancer tissue was handled within 3 h after excision from the patient. Briefly, the tissue was cut into approximately 1 mm³ pieces in DMEM, and then enzymatically treated with MACS tumour dissociation kit (Miltenyi Biotec, Cat. 130‐095‐929) using 37C_h_TDK_3 program in the gentleMACS Octo Dissociator with Heaters. Dissociated cells, while re‐suspended in DMEM, were subsequently filtered through a 70 μm cell strainer (BD) and centrifuged at 400 g for 10 min at 4°C. After removing the supernatant, the cell pellet was re‐suspended by 1X phosphate‐buffered saline (PBS) with 10% FBS, and the red blood cells were removed using the red blood cell lysis buffer (Roche), according to the manufacturer's instructions. Cells were then filtered through a 40 μm cell strainer (BD), and single cells were picked up by mouth pipetting for scGTP‐seq.