GSM9385061: AKPS_Helicobacter negative_day 21_3 S4; Mus musculus; RNA-Seq
Tumors were minced and digested in digestion medium consisting of RPMI1640 (Corning) supplemented with 10% FBS, collagenase D (2.5mg/ml; Sigma-Aldrich), Liberase DL (0.5mg/ml; Sigma-Aldrich), and DNase I (0.2mg/ml; Sigma-Aldrich). Digestion was carried out at 37°C with rotation at a slow speed (25 rpm ) for 30-60 minutes. Digested samples were filtered through 100-µm cell strainers, and remaining tissue fragments were mechanically disrupted using plunger of a syringe. Cells were centrifuged at 300 x g for 5 minutes, washed once with RPMI1640, and stained with CD45-APC/Cy7 (immune cells) or EpCAM-APC (epithelial cells) antibodies (1:200, BioLegend). CD45+ EpCAM- cells and CD45- EpCAM+ cells were sorted using Sony SH800S cell sorter and combined at an 80:20 ratio, followed by single cell library preparation. Single cell libraries were generated using a Chromium Controller (10X Genomics) using Chromium Next GEM single cell 3' Gene Expression kit (10X Genomics), according to manufacturer's instructions at CSHL Single Cell Biology Shared Resource. Cell suspensions were loaded to target 8000 cells per sample. cDNA and libraries were assessed using Agilent 2100 Bioanalyzer, quantified by KAPA qPCR, and sequenced on either a NextSeq500 or Novaseq2000 (Illumina) to a depth of approximately 20.000 reads per cell. FASTQs files aligned to the mouse reference genome (gex-mm10-2020-A) and digital gene-cell count matrices were generated using default parameters.