title: scGET-seq profile of patient derived organoids from colorectal cancer samples
design: scGET-seq profile of patient derived organoids from colorectal cancer samples
library_construction_protocol: 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.