Single cell RNA-seq profiles of patient derived organoids
Single cell RNA-seq profiles of patient derived organoids
Fresh tumor specimens obtained from patients with liver metastatic gastrointestinal cancers were used immediately after surgery. Tissues were minced, conditioned in PBS/5mM EDTA and digested in a solution composed of PBS/1mM EDTA, 2X TrypLE™ Select Enzyme and DNAse I for 1h at 37°C. Dissociated cells were collected, resuspended in 120μl growth factor reduced Matrigel™, seeded in single domes in 24-well flat bottom cell culture plate and, after dome solidification, overlaid with 1ml of complete human organoid medium and medium replaced every two/three days. PDOs were dissociated to single cells either for passaging after reaching confluence or for the subsequent downstream applications by mechanical and enzymatic digestion. PDOs were retrieved from Matrigel™ in a solution composed of PBS/1mM EDTA and 1X TrypLE™ Select Enzyme, incubated for 20 min at 37 °C then dissociated to single cells by pipetting.  Cells were harvested, resuspended in growth factor reduced Matrigel™ Nucleic acids were directly processed on intact cells following standard Chromium Single Cell 3' Reagent v3 chemistry protocol (10X Genomics) using  Chromium platform (10x Genomics). Target cell recovery was 5'000. Libraries were prepared using Chromium Single Cell 3' Reagent v3 chemistry protocol (10X Genomics). Library quality was assessed using TapeStation (Agilent Technologies). Libraries were sequenced on NovaSeq 6000 platform (Illumina) aiming at 50'000 clusters/cell.