characteristics.text: ERC000011
characteristics.characteristic: ENA-CHECKLIST
characteristics.text: 2020-10-02T04:04:59Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2019-10-08T11:36:34Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA6059248
characteristics.characteristic: External Id
characteristics.text: Istituto Nazionale Genetica Molecolare INGM ‘Romeo ed Enrica Invernizzi’, Milan 20122, Italy.
characteristics.characteristic: INSDC center name
characteristics.text: 2020-10-02T04:04:59Z
characteristics.characteristic: INSDC first public
characteristics.text: 2019-10-08T11:36:34Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS3860377
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-8416:SQ_2355
characteristics.characteristic: Submitter Id
characteristics.text: 82
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: SQ_2357
characteristics.characteristic: associate input name
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: Primary colonic tissues were obtained from the hospital. Normal and tumoral tissues were processed according to Fujii et al (Fujii et al, 2015) with few modifications. Surgically resected specimens were reduced to a size of 3-5 mm, extensively washed with cold PBS and gentamicin (20microg/ml) and incubated with PBS-EDTA (2,5mM) rocking on a wheel for 1h at 4°C. After PBS-EDTA tissues samples were further washed with cold PBS and normal crypts and tumoral cells were released and collected by centrifuging at 400g 5’ 4°C. Patient-derived colorectal cancer organoids (PDOs) were established and maintained as previously described (Fujii et al 2015; Van De Wetering et al 2015; Sato et al 2011) with few modifications. Tumor suspension cells isolated from human colorectal cancer biopsies were embedded in Matrigel® Growth Factor Reduced Basement Membrane Matrix, Phenol Red-Free (Corning), passaged once per week. PDOs were maintained in 24 well plate overlaid by the organoid culture medium (Advanced DMEM/F12 (Life Technologies) supplemented with penicillin/streptomycin, 10mM HEPES (Life Technologies), 2mM GlutaMAX (Life Technologies), 1X B27 (Life Technologies), 1x N2 (Life Technologies), 1 mM N-Acetyl Cysteine (Wako), 10 mM Nicotinamide (Sigma), with the following growth factors, 50 ng/ml human EGF (Peprotech), 100 ng/ml human Noggin (Peprotech), 10 nM Gastrin (Sigma), 500 nM A83-01 (Tocris), 10 μM SB202190 (Sigma)). ChIPmentation was carried out as previously described (Schmidl et al., 2015), with the following modifications. Briefly, crosslinked organoids were incubated 30 minutes in lysis buffer 1 (50mM HEPES, pH7.5, 10mM NaCl, 1mM EDTA, 10% Glycerol, 0.5% NP-40, 0.25% Triton X-100, plus protease inhibitors) at 4°C. Organoids were recovered by centrifuging 800g 5’ at 4°C in 1,5 ml tubes and lysed in buffer 2 (10mM Tris-HCl pH8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA, plus protease inhibitors) for 10 minutes at room temperature. Organoids pellet was sonicated in lysis buffer 3 (10mM Tris-HCl pH8, 200 mM NaCl, 1mM EDTA, 0.5 mM EGTA, 0.1% Na-deoxyxholate, 0.5% N-lauroysarcosine, plus protease inhibitors) using Covaris® M220 focused-ultrasonicator (duty factor 10%, peak incidence power 75 Watt, cycles per burst 200, 8/15 minutes). Sonicated chromatin (∼ 7 µg) was incubated with anti-WWTR1 (0,4 µg - Sigma Aldrich, HPA007415), anti-YAP1 (abcam 52771) or anti-rabbit IgG (SinoBiological CR1) overnight at 4 °C on the wheel. Antibody/antigen complexes were recovered with blocked Protein G-Dynabeads (Life Technologies) for 2 h at 4 °C and washed twice with low salt wash buffer ((20mM Tris-HCl pH8, 150mM NaCl, 0.1 % SDS, 1% Triton X-100 and 2mM EDTA), twice with high salt buffer (20mM Tris-HCl pH8, 2mM EDTA, 500mM NaCl, 0.1% SDS and 1% TritonX) and once with 10mM Tris pH 8.0. Washed immunocomplexes were resuspended in tagmentation reaction containing 0,5 μl Tagment DNA Enzyme from Nextera DNA Sample Prep Kit (Illumina) and incubated at 37 °C for 10 min. Beads were washed with low-salt buffer, twice with 1XTE and incubated with ChIP elution buffer (10mM Tris pH 8.0; 5mM EDTA; 300mM NaCl; 0,4% SDS) plus 0,8 mg/ml Proteinase K (NEB) for 1 h at 55 °C and 8 h at 65 °C, to revert formaldehyde cross-linking. The immunoprecipitated and input DNA were then purified by Qiagen MinElute kit (Qiagen) and eluted in 22 μl EB buffer. ChIPmentation-seq libraries were prepared using custom Nextera primers as described in Buenrostro et al., (2013) and enriched libraries were purified using 1,8V of SPRI AMPure XP beads.
characteristics.characteristic: description
characteristics.text: late
characteristics.characteristic: developmental stage
characteristics.text: colorectal cancer
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005842
characteristics.characteristic: disease
characteristics.text: organoid culture
characteristics.characteristic: growth condition
characteristics.text: 10
characteristics.characteristic: individual
characteristics.text: Homo sapiens
characteristics.characteristic: organism
characteristics.text: Homo sapiens
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: organism
characteristics.text: colon
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0001155
characteristics.characteristic: organism part
characteristics.text: tumor tissue
characteristics.characteristic: sampling site
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: SQ_2355
characteristics.characteristic: title