characteristics.text: 2013-06-12T17:02:04Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:29:39Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA2077999
characteristics.characteristic: External Id
characteristics.text: Institute for Molecular Biology and Tumor Research
characteristics.characteristic: INSDC center name
characteristics.text: 2013-06-12T17:02:04Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:29:39Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS254535
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1683:CKI alpha Het
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: epithelial cell
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000066
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: For generation of conditional CKI knockout mice, Cre was transiently expressed in ES cells to produce specific deletion of the neomycin cassette and an intact floxed CKI alpha allele. A second aggregation was done, and Vilin-Cre-ERT2 transgenic mice were used as a gut-specific deleter to generate a conditional CKI floxed/floxed Villin-Cre-ERT2 knockout mouse (CKI alpha KO). Inducible double-knockout mice (p53 and CKI alpha) were generated by crossing mice in which CKI alpha was floxed with mice in which p53 was floxed (CKI alpha/p53 DKO). Mice were kept under specific pathogen-free conditions at the Hadassah Medical School of the Hebrew University. Mice were injected with 150 milligram/kilogram Tamoxifen (Sigma) subcutaneously, at a regimen of 6 injections every other day. Sulindac (Sigma) was administered in the drinking water at 0.6 gram/liter, one week before the first Tamoxifen injection, and throughout the 2 weeks of knockout induction, for a total period of 3 weeks. BV6 (Genentech) was injected intraperitoneally at 10 milligram/kilogram, every other day, from day 2 of knockout induction. BOT64 (Santa Cruz) was injected intraperitoneally at 30 milligram/kilogram, every day, from day 2 of knockout induction. 1-2 days after the last Tamoxifen injection, mice were injected intraperitoneally with 10 microliter/gram of 5-bromo-2-deoxyuridine (BrdU; GE Healthcare) and sacrificed 2 hours later, on day 10-12 of knockout induction. Jejunum, ileum and the entire large intestine were flushed with ice-cold PBS, cut open longitudinally, and subjected to fixation in 4% formaldehyde and paraffin embedding(FFPE). Intestinal epithelial cells (IECs) were isolated from the middle part of the small intestine and separated into single cells in HBSS containing 10 millimolar HEPES, 5 millimolar EDTA and 0.5 mM DTT, at 37C for 30 minutes. Total RNA was extracted from IECs using TRI-reagent (Sigma) and phenol/chloroform methods. Total RNA from IECs of three mice per genotype was pooled, processed with the mRNA Sequencing Kit (Illumina). Library construction was done with the Illumina TRUSeq kit according maufacturers instructions.
characteristics.characteristic: description
characteristics.text: flox sites around one CKI alpha allele
characteristics.characteristic: genomic modification
characteristics.text: cki alpha -/+
characteristics.characteristic: genotype
characteristics.text: Mus musculus
characteristics.characteristic: organism
characteristics.text: Mus musculus
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_10090
characteristics.characteristic: organism
characteristics.text: intestine
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/UBERON_0000160
characteristics.characteristic: organism part
characteristics.text: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: CKI alpha Het
characteristics.characteristic: title