GSM6576367
Within 6 hours after isolation, solid specimens were enzymatically dissociated into single cells. Briefly, the tissue was minced with a scalpel and enzymatically digested using 2 mg/mL Collagenase I (Worthington Biochemical) and 2 mg/mL Collagenase IV (Worthington Biochemical) for 30 minutes in a shaker (250 rpm) at 37°C. The digestion was terminated with DMEM + 5% fetal bovine serum (Thermo Fisher Scientific). The cell suspensions were sequentially filtered through 100 μm and then 70 μm cell strainers. Red blood cells were lysed by incubating the cell suspensions in RBC Lysis Solution (Sigma- Aldrich) for 3–10 minutes at 4°C. After centrifugation and resuspension, the concentrations of the single-cell suspensions were adjusted to 7–12×105 cells/ml with 5% fetal bovine serum DMEM. Ascites was centrifuged for 10 min at 4°C, and the remaining pellet was resuspended in PBS, filtered, subjected to RBC lysis, and resuspended as described for the tumor samples. Peripheric blood was collected into heparin tubes (Becton, Dickinson and Co.) and processed within 2 hours of collection. PBMCs were isolated by density gradient centrifugation using Ficoll-Paque Plus medium and washed with Ca/Mg-free PBS. The isolated cells derived from above samples were used for further single cell sequencing. The suspensions of live cells in sterile-filtered PBS (Corning) with 0.04% BSA (Sigma Aldrich) were used as input for the 10× Chromium controller system (10× Genomics Inc.). Using 10× GemCode Technology, the cells were barcoded to separately index each cell's transcriptome by partitioning oil and gel beads to generate the Gel Bead-in-EMulsions (GEMs). Barcoded Single Cell 50 Gel Beads, RT Master Mix with cells and Partitioning Oil were combined on a microfluidic chip, and GEMs were generated. The GEM RT reactions were activated in a thermocycler (53°C for 45 min, 85°C for 5 min, 4°C hold overnight). Post RT incubation, the GEMs were disrupted and the first-strand cDNA was recovered. cDNA amplification was performed by PCR to generate sufficient material. According to the manufacturer's instructions, scRNA-seq libraries of tumor tissue and ascites were generated using Chromium™ Single Cell 3' Library (v3 chemistry) reagents. For PBMC samples, scRNA-seq libraries were processed using the Chromium™ single cell 5' library & gel bead kit and coupled TCR/BCR libraries were obtained using the Chromium™ single cell V(D)J enrichment kit (10× Genomics). Libraries of scRNA-seq were sequenced on the Illumina Novaseq 6000.