title: HCT116_Dicer_EX5_nuclear_input
organism: Homo sapiens
description: Protocols: HCT 116 WT and DICER_Ex5 cells were grown in McCoy's 5A medium supplemented with 10% (v/v) fetal bovine serum, 2mM L-Glutamine and Penicillin-Streptomycin. Nuclei of 350 x 10E+6 HCT116 WT and DICER_Ex5 were lysed in IP-buffer supplemented with RNasin (Promega). Nuclear lysates were clarified and pre-cleared in the presence of Protein G-sepharose beads for 2 hr 4 degrees C under constant rotation and then filtered through a 0.45 um filter. Lysate aliquots were taken out as Input for RNA and protein isolation. Antibody anti-AGO2 (11A9, Ascenion) and isotype-matched IgG (mock_IP) were coupled to Protein G-sepharose beads for 2 h at 4 degrees C in IP-buffer containing 1 mg/ml heparin (Sigma). The pre-cleared nuclear lysate was incubated with antibody-coupled beads overnight at 4 degrees C under constant rotation. IP samples were washed once with IP-buffer for 5 min at 4 degrees C, and three times with IP-wash buffer (50 mM Tris-HCl, pH 7.5; 300 mM NaCl; 5 mM MgCl2; and 0.05% Nonidet P-40) at 4 degrees C. An aliquot was taken out for western blot in order to check immunoprecipitation efficiency. For RNA isolation, Input and IP samples were DNAseI- and Proteinase K-treated. Total RNA and co-precipitated RNA was extracted by phenol:chloroform:isopropyl alcohol and precipitated in ethanol. Small RNA libraries were constructed using the TruSeq SmallRNA Sample preparation kit (Illumina)
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2014-03-03T16:32:10Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2018-03-08T16:53:32Z
attributes.tag: External Id
attributes.value: SAMEA2242748
attributes.tag: INSDC center name
attributes.value: La Sapienza' University of Rome
attributes.tag: INSDC first public
attributes.value: 2014-03-03T16:32:10Z
attributes.tag: INSDC last update
attributes.value: 2018-03-08T16:53:32Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-2056:HCT116_Dicer_EX5_nuclear_input
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: HCT116_dicer_Ex5
attributes.tag: common name
attributes.value: human
attributes.tag: genetic modification
attributes.value: targeted mutation
attributes.tag: genotype
attributes.value: Dicer hypomorphic
attributes.tag: sample name
attributes.value: E-MTAB-2056:HCT116_Dicer_EX5_nuclear_input
attributes.tag: scientific_name
attributes.value: Homo sapiens
attributes.tag: specimen with known storage state
attributes.value: frozen specimen