scGET-seq profile of patient derived organoids from colorectal cancer samples
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 permeabilized nuclei following a modification of the standard ATAC-seq protocol (doi: 10.1002/0471142727.mb2129s109) detailed in “Nuclei Isolation for Single Cell ATAC Sequencing” protocol CG000169, Rev D (10X Genomics). Target nuclei recovery was  5'000 nuclei Single-cell ATAC-seq and GET-seq were performed on Chromium platform (10x Genomics) following “Chromium Single Cell ATAC Reagent Kits” user manual version CG000168 Rev C. For GET-seq protocol, Tn5 and TnH were used in substitution of the 10X ATAC enzyme (10X Genomics) in the transposition mix assembly step. When a sequential Tn5 to TnH reaction was performed, a transposition mix containing 1.5 uL of Tn5 was incubated 30 min at 37 °C, then 1.5 uL of TnH was added and the reaction was continued for a total of 1 h incubation.