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: SAMEA6059240
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: ERS3860369
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-8416:SQ_2297
characteristics.characteristic: Submitter Id
characteristics.text: 58
characteristics.unit: year
characteristics.characteristic: age
characteristics.text: SQ_2299
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. ∼0,3x10^6 organoid cells/well, normal crypts and tumoral cells pellets were fixed in Formaldheyde - PBS solution (final 1%), for 10 minutes rocking at room temperature and quenched with 0,125M Glycine for 5 minutes. PBS-washed organoid pellets were lysed in 500μl of 1X sonication lysis buffer (10 mM Tris pH 8.0, 0.25% SDS, 2 mM EDTA, plus protease inhibitors). Lysed chromatin was sheared using Covaris® M220 focused-ultrasonicator (settings: duty factor 20%, peak incidence power 75 Watt, cycles per burst 200, 8/15 minutes). ∼500/1000 ng of sonicated chromatin was incubated with 0,5/1μg amount of the indicated antibodies (H3K27Ac abcam 4729; H3K4me3 Millipore 07-473; H3K4Me1 DIAGENODE C15410194; H3K36me3 DIAGENODE C15410192; H3K27me3 07449 Millipore), overnight at 4 °C. Immunocomplexes were recovered with blocked 10 μl Protein G-Dynabeads (Thermo Fisher) for 2 h at 4 °C and washed twice with RIPA-low salt (10mM Tris-HCl pH 8.0, 140mM NaCl,1mM EDTA pH 8.0, 0.1% SDS and 0.1% Na-Deoxycholate,1% Triton x-100), twice with RIPA-high salt (10mM Tris-HCl pH 8.0, 1mM EDTA pH 8.0, 500mM NaCl, 1% Triton x-100, 0.1% SDS, 0.1% DOC) , twice with RIPA-LiCl (10mM Tris-HCl, pH 8.0, 1mM EDTA, pH 8.0, 250mM LiCl, 0.5% NP-40, 0.5% DOC), once with 10mM Tris pH8.0 and once with 1X TE, followed by reverse crosslinking overnight. The immunoprecipitated DNA and input were then purified by Qiagen MinElute kit (Qiagen) and eluted in 22 μl EB buffer. ChIP-seq libraries were prepared according to TruSeq ChIP Library Preparation Kit from Illumina.
characteristics.characteristic: description
characteristics.text: colorectal cancer
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0005842
characteristics.characteristic: disease
characteristics.text: 41
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: healthy adjacent tissue
characteristics.characteristic: sampling site
characteristics.text: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: male
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000384
characteristics.characteristic: sex
characteristics.text: SQ_2297
characteristics.characteristic: title