organism: Mus musculus
description: Protocols: Ileum samples were flushed with ice-cold PBS and enterocytes were extracted by shaking them in tissue lysis buffer (HBSS, 5 Mm EDTA and 250 µM DTT) for 30 minutes at 37oC. Mice were kept under specific pathogen-free conditions at the Hadassah Medical School of the Hebrew University. All mouse experiments were performed in accordance with guidelines of the relevant institution's ethics committee. CKIα and p53 KO was induced by 4 subcutaneous tamoxifen injections (150 mg/kg) every alternate day, followed by mice sacrifice. Freshly extracted enterocytes were fixed and cross-linked using 1% paraformaldehyde solution for 10 minutes and neutralized with 125 mM glycine for 5 minutes. Cells were resuspended and lysed in RIPA buffer containing 0.5% SDS. To solubilize and shear cross-linked DNAs, lysates were sonicated with Covaris M220 Sonicator at 75W with 20% duty factor, 200 cycles for 20 minutes. Sonicated lysates were centrifuged at 13,000 RPM for 5 minutes and the chromatin was incubated overnight at 4oC with immune-beads. Following ChIP (using the Rabbit polyclonal p53 FL-393 antibody from Santa Cruz Biotechnology, catalog number SC-6243ac), samples were washed and bound complexes were eluted by Direct Elution Buffer (10 mM Tris-HCl pH 8, 0.3 M NaCl, 5 mM EDTA and 0.5% SDS), followed by treatment with RNase A (ThermoFisher Scientific) and Proteinase K (Invitrogen). Chromatin was purified with QIAquick Gel Extraction Kit (QIAGEN). ChIP-seq libraries were prepared from purified ChIP DNA with the Microplex Library Preparation Kit (Diagenode) according to the manufacturer's instructions.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2020-01-03T15:59:19Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-04-08T10:00:11Z
attributes.tag: External Id
attributes.value: SAMEA5555911
attributes.tag: INSDC center name
attributes.value: Philipps-University Marburg, Institute of Molecular Oncology and Genomics Core Facility, Center for Tumor and Immunobiology
attributes.tag: INSDC first public
attributes.value: 2020-01-03T15:59:19Z
attributes.tag: INSDC last update
attributes.value: 2019-04-08T10:00:11Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7858:CKIa_p53_DKO
attributes.tag: age
attributes.value: 3
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: genotype
attributes.value: CKIgut/p53gut
attributes.tag: individual
attributes.value: pool of mice
attributes.tag: organism part
attributes.value: ileum
attributes.tag: sample name
attributes.value: E-MTAB-7858:CKIa_p53_DKO
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: C57BL/6