2024-06-03
ENA first public
ERC000011
ENA-CHECKLIST
SAMEA113994444
External Id
Center for Omics Sciences, IRCCS San Raffaele Hospital
INSDC center name
2023-07-06T12:08:33Z
INSDC last update
public
INSDC status
ERS15978291
SRA accession
E-MTAB-13126_3:Sample 1
Submitter Id
ArrayExpress
broker name
not collected
collection date
human
common name
Protocols: 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.
description
adult
developmental stage
colorectal cancer
disease
wild type genotype
genotype
not collected
geographic location (country and/or sea)
organoid
growth condition
not applicable
isolate
liver
metastatic site
Homo sapiens
organism
Homo sapiens
organism
liver
organism part
neoplasm
sampling site
Homo sapiens
scientific_name
Sample 1
title