GSM5101020: Pt3_deep_cancer; Homo sapiens; RNA-Seq
The single cells were isolated from fresh tumor tissues and paired normal gastric tissues. The tissues were cut to small pieces and minced with scalpels in a culture dish. Small pieces were washed with PBS and transfer the tissue pieces into the gentleMACS C tubes (Macs, CA, USA) and tumor dissociated using a Human Tumor Dissociation Kit (Miltenyi Biotec, Bergisch Gladbach, Germany). Cell suspensions were filtered using a 100μm filter, and dissociated cells were pelleted and resuspended in PBS. Then we performed dead cell remove in single cell using a Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) per manufacturer guidelines. The 10x Genomics Chromium platform was used to capture and barcode the cells to generate single-cell Gel Beads-in-Emulsion (GEMs) by following the manufacturer's protocol. Briefly, along with the reverse transcription master mix, cell suspensions were loaded onto 10x Genomics Single Cell 30 Chips. During this step, cells were partitioned into the GEMs along with gel beads coated with oligonucleotides. These oligonucleotides enable mRNA capture inside the droplets by 30 bp oligo-dT after cell lysis and provide barcodes to index cells (16 bp) as well as transcripts (10 bp UMI). Following reverse transcription, cDNAs with both barcodes were amplified, and a library was constructed using the Single Cell 30 Reagent Kit (v2 chemistry) for each sample. The resulting libraries were sequenced on an Illumina HiSeq4000 System in a 2 × 100 bp paired-end mode.