Illumina Genome Analyzer II sequencing; RNA CLIP-seq for Cugbp1, Mbnl1 and Ptbp1 in mouse C2C12 myoblasts
RNA CLIP-seq for Cugbp1, Mbnl1 and Ptbp1 in mouse C2C12 myoblasts
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
Experimental Factor: IMMUNOPRECIPITATE
Cugbp1