characteristics.text: 2012-01-04T17:19:56Z
characteristics.characteristic: ENA-FIRST-PUBLIC
characteristics.text: 2018-03-08T15:27:40Z
characteristics.characteristic: ENA-LAST-UPDATE
characteristics.text: SAMEA1032097
characteristics.characteristic: External Id
characteristics.text: SDU
characteristics.characteristic: INSDC center name
characteristics.text: 2012-01-04T17:19:56Z
characteristics.characteristic: INSDC first public
characteristics.text: 2018-03-08T15:27:40Z
characteristics.characteristic: INSDC last update
characteristics.text: public
characteristics.characteristic: INSDC status
characteristics.text: ERS042757
characteristics.characteristic: SRA accession
characteristics.text: E-MTAB-414_2:Ptbp1
characteristics.characteristic: Submitter Id
characteristics.text: ArrayExpress
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0009737
characteristics.characteristic: broker name
characteristics.text: C2C12
characteristics.ontologyTerms: http://www.ebi.ac.uk/efo/EFO_0001098
characteristics.characteristic: cell line
characteristics.text: myoblast
characteristics.ontologyTerms: http://purl.obolibrary.org/obo/CL_0000056
characteristics.characteristic: cell type
characteristics.text: house mouse
characteristics.characteristic: common name
characteristics.text: Protocols: DMEM with 10% fetal bovine serum at 37C with 5% CO2. Antibodies to Cygbp1 (3B1) and Ptbp1 (N20) were purchased from Santa Cruz Biotechnology. Anti-Mbnl1 rabbit serum (A2764) was a kind gift from Dr. Charles A. Thornton at University of Rochester. C2C12 cells were UV-irradiated at 400 mJ and whole-cell lysates were prepared with lysis buffer (50 nM Tris-HCl pH 7.4, 100mM NaCl, 1mM MgCl2, 0.1mM CaCl2, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS). The extracts were treated with 30 uL of RNase-free DNaseI (Qiagen) for 5 min at 37C, and subsequently treated with dilute RNaseA (Ambion) at a final dilution of 1:1000 for 10 min at 37C. After centrifugation at 14,000 rpm for 20 min at 4C, 2 ug of specific antibody was added to the supernatant and incubated at 4C. After 2 hours, 20 uL of Dynabeads protein G was added to the lysate and incubated for 1 hour at 4C. Then, beads were washed 6 times and the trimmed input RNA on the beads was ligated to the 3' RNA linker at 15C overnight. Beads were then washed and incubated with polynucleotide kinase and radioactive ATP to label the cross-linked RNA. The protein-RNA complexes were separated by NuPAGE (Invitrogen) and electroeluted. The electroeluate was digested with 4 mg/mL proteinase K, and the RNA was recovered by acidic phenol/chloroform extraction and ethanol precipitation. The recovered RNA was ligated to the 5' RNA linker at 15C for 8 hours and was recovered by acidic phenol/chloroform extraction and ethanol precipitation after DNaseI treatment. RNA was reverse-transcribed and PCR-amplified. Gel-purified amplicons from the primary RT-PCR were reamplified for 10-15 cycles, and were purified after Exo-SAP (USB) treatment. Quality and quantity of PCR products were analyzed by the Agilent 2100 Bioanalyzer. Library fragment size: 70-150bp
characteristics.characteristic: description
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: Mus musculus
characteristics.characteristic: scientific_name
characteristics.text: Ptbp1
characteristics.characteristic: title