title: Illumina Genome Analyzer II sequencing; IMP2/IGF2BP2 HITS-CLIP from E13.5 mouse brain
design: IMP2/IGF2BP2 HITS-CLIP from E13.5 mouse brain
library_construction_protocol: Brain tissues were dissected from E13.5 Swiss Webster mouse brains. CLIP experiments were performed with E14 Swiss Webster mouse brains as described (Ule 2003; Ule 2005; Licatalosi 2008; Preitner 2014). Briefly, embryonic brain tissues were partially dissociated before UV irradiation (UV at 254 nm, 3X 300 mJ/cm2 in a Stratalinker). Cell lysates were sonicated, treated with DNase, and digested with benzonase (Sigma E8263; at a final dilution of 1:2000; 10 min at 37°C) to produce protein-bound RNAs of ~40 nt fragments. Then pre-cleared lysates were mixed with 30 μl Sepharose beads and 30 μg antibody (mouse polyclonal anti-IMP2 antibody, Abnova H00010644-A01; rabbit anti-IMP2 antibody, Proteintech 11601-1-AP), and rotated for 3 hours at 4°C. Immunoprecipitated protein-RNA complexes were stringently washed with SDS- containing buffer and dephosphorylated, and the radiolabeled RNA adapter (5’- UCGUAUGCCGUCUUCUGCUUGU-3’) were ligated at the 3’ end of the RNA fragments. Protein-RNA complexes were then phosphorylated at the 5’ end, separated on a 4-12% denaturing NuPAGE gel (Invitrogen), transferred to a nitrocellulose membrane, and exposed on a film. Narrow bands corresponding to the size of the specific protein-RNA complexes were then excised from the membrane. RNA fragments were extracted, ligated to the 5’ adapter (5’-GUUCAGAGUUCUACAGUCCGACGAUC-3’) and amplified by RT-PCR (primers: AATGATACGGCGACCACCGACAGGTTCAGAGTTCTACAGTCCGA, and CAAGCAGAAGACGGCATACGA). The purified PCR products were sequenced on Illumina sequencing platform.
attributes.tag: Experimental Factor: IMP2 antibody 1
attributes.value: immunoprecipitate