GSM6604053: TLE_HS_5 (Ago iCLIP); Homo sapiens; OTHER
Hippocampal tissue is triturated and crosslinked by 254 nm UV. Then cells are lysed and partially digested with RNase I. Ago immuno-precipitation is performed on the cell lysate with antibody coupled to Protein A dynabeads. RNA, still attached to antibody, is 3' linker ligated and 5' end labeled with 32P-γ-ATP. RNA is eluted from the beads, run on SDS-PAGE and transferred to a nitro-cellulose membrane where the Ago-RNA bands are cut out and treated with Proteinase K to remove Ago protein. After RNA purification, RT-PCR and gel purification, the RNA is circularized by use of the CircLigase II enzyme. BamHI digestion lineralizes the RNA and after PCR amplification, the finished Ago iCLIP library is formed.