Homo sapiens
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)
ENA-FIRST-PUBLIC
2014-03-03T16:32:10Z
ENA-LAST-UPDATE
2018-03-08T16:53:32Z
External Id
SAMEA2242749
INSDC center name
La Sapienza' University of Rome
INSDC first public
2014-03-03T16:32:10Z
INSDC last update
2018-03-08T16:53:32Z
INSDC status
public
Submitter Id
E-MTAB-2056:HCT116_Dicer_EX5_cells_RIP_anti-IgG
broker name
ArrayExpress
cell line
HCT116_dicer_Ex5
common name
human
genetic modification
targeted mutation
genotype
Dicer hypomorphic
sample name
E-MTAB-2056:HCT116_Dicer_EX5_cells_RIP_anti-IgG
scientific_name
Homo sapiens
specimen with known storage state
frozen specimen