GSM8730349: P29_pre_TCR; Homo sapiens; RNA-Seq
We collected fresh tumor tissues on the day of surgery, which were mechanically and enzymatically dissociated using a gentleMACS dissociator (Miltenyi Biotec, Gladbach Bergisch, Germany, Cat#130-093-235) and the Human Tumor Dissociation Kit (Miltenyi Biotec, Cat#130-095-929), following the manufacturer's instructions. After incubation for 1 hour at 37℃, the resuspended samples were filtered through a 70-µm MACS SmartStrainer (Miltenyi Biotec, Cat#130-098-462) into RPMI-1640 medium (Corning, Inc., Corning, NY, USA) supplemented with 10% fetal bovine serum (Biowest, Riverside, MO, USA) and centrifuged at 300 × g for 10 minutes. The pellet was suspended in PBS containing 2% fetal bovine serum. Tissue-infiltrating lymphocytes were enriched using a Ficoll gradient (Sigma-Aldrich), and the resulting single-cell suspensions were subsequently counted using trypan blue. Libraries were constructed using the Chromium controller, adhering to the protocol described in the 10x Chromium Next GEM Single Cell 5′ v2 Cell Surface Protein User Guide (CG000330). Initially, cell suspensions were diluted with nuclease-free water to achieve a target cell count of 10,000. These suspensions were then combined with a master mix and loaded with Single Cell 5′ Gel Beads and Partitioning Oil into a Next GEM Chip K. Within the droplets, RNA transcripts from individual cells were uniquely barcoded and reverse-transcribed. Following this, the cDNA molecules were pooled and subjected to PCR enrichment. The amplified cDNA underwent size selection for the 5′ Gene Expression libraries, V(D)J Enriched Libraries, and Cell Surface Protein libraries. For the Cell Surface Protein Library, the cDNA pool was enriched through index PCR. In the case of the V(D)J Enriched Library, the enriched cDNA pool was amplified through two rounds using specific primers. For the 5′ Gene Expression Library, the cDNA pool underwent end repair, addition of a single 'A' base, and ligation of adapters, followed by purification and PCR enrichment. The purified libraries were quantified using qPCR in accordance with the qPCR Quantification Protocol Guide (KAPA) and assessed for quality using the Agilent Technologies 4200 TapeStation. Finally, the libraries were sequenced on the HiSeq platform (Illumina) following the specified read length outlined in the user guide.