GSM2151619: Mouse brain tissue_rep2; Mus musculus; OTHER
Adult female was sacrificed by cervical dislocation and the whole brain was immediately collected, rinsed with ice-cold PBS three times and snap frozen. Frozen whole mouse brain tissue was ground into fine powder in liquid nitrogen using a mortar and pestle. The tissue powder was quickly transferred into a Petri dish on a bed of dry ice and irradiated on dry ice three times at 400 mJ/cm2 in a UV crosslinker (254 nm) with gentle swirling between each irradiation. Crosslinked powdered tissue was immediately lysed and subjected to MARIO procedure as described. Protein-RNA complex was eluted from streptavidin beads and RNA is recovered by digesting the bound protein. Eluted RNA was subjected to rigorous DNase treatment to eliminate DNA contamination. Purified RNAs were hybridized with a DNA probe that is complementary to the biotin-tagged RNA linker, and treated with T7 exonuclease to remove the non-ligated biotinylated RNA linkers. As a result, mainly the successfully ligated chimeric RNAs retained a biotin-tagged linker at the junction. This chimeric RNA library was fragmented again to an average of 150 nucleotides, and the ligation junctions were pulled-down with streptavidin-coated magnetic beads. The end product was a library of ~150nt chimeric RNAs. This library was enriched with chimeras of in the form of 5'-R1-linker-R2, where R1 and R2 were fragments of interacting RNAs. This library was converted into cDNAs and sequenced with paired-end next-generation sequencing.
GEO Accession
GSM2151619