characteristics.text: 2013-01-31T17:01:38Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T16:08:42Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1695734
characteristics.characteristic: External Id
characteristics.text: EMBL-EBI
characteristics.characteristic: INSDC center name
characteristics.text: 2013-01-31T17:01:38Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T16:08:42Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS193712
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-1371:U2AF65 KD1 replicate 2 (LUjh21_2)
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: HeLa
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001185
characteristics.characteristic: cell line
characteristics.text: human
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/NCBITaxon_9606
characteristics.characteristic: common name
characteristics.text: Protocols: HeLa cells were grown in Dulbecco's modified Eagle medium (DMEM) supplemented with 10% Fetal Bovine Serum and 1% penicillin-streptomycin and cultured at 37 C with 5% CO2. For the knockdown of HNRNPC, HeLa cells were plated at concentration of 105 cells/well in a 6-well cluster plate in 2 ml of growth medium. 24 h after the plating (when confluency reached around 20%), cultures were independently transfected using two different hnRNP C Stealth Select RNAi siRNAs (KD1 and KD2 refer to siRNAs HSS179304 and HSS179305 from Invitrogen, respectively) at a final concentration of 5 nM. Both siRNAs were transfected using Lipofectamine RNAiMAX (Invitrogen) according to the manufacturers instructions. Using the same procedure, we used Stealth RNAi siRNA Negative Control (Invitrogen) for the control experiments. Protein extracts were prepared from UV-crosslinked (UV+) control HeLa cells (Ctrl) and HNRNPC knockdown cells (KD1 and KD2), and RNA was partially digested using low or high concentrations of RNase. RBP-RNA complexes were immuno-purified with a mouse antibody against hnRNP C or U2AF65. To allow visualization of the protein-RNA complexes, the 5' ends of the RNAs were radioactively labeled. The complexes were size-separated using denaturing gel electrophoresis and transferred to a nitrocellulose membrane. RNA recovered by proteinase K digestion from the nitrocellulose membrane was reverse transcribed and size-selected using denaturing gel electrophoresis. Three of the cDNA size fractions (H: 100-170 nt; M: 85-100 nt; L: 75-85 nt) were further processed and PCR amplified (17 cycles of amplification) to obtain the iCLIP libraries.
characteristics.characteristic: description
characteristics.text: HNRNPC knockdown (KD1)
characteristics.characteristic: genetic modification
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: Homo sapiens
characteristics.characteristic: scientific_name
characteristics.text: female
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/PATO_0000383
characteristics.characteristic: sex
characteristics.text: U2AF65 KD1 replicate 2 (LUjh21_2)
characteristics.characteristic: title