GSM7139760: Rep2 Ded1-RNA X-link grown with glucose depletion; Saccharomyces cerevisiae; RIP-Seq
Cells were lysed by vortexing in a FastPrep-24, clarified by centrifugation, and then bound to Protein A-Sepharose beads with Ded1 IgG, washed extensively and digested with nuclease S1 The 3' adapter was added directly to the Protein-Sepharose-bound Ded1-RNA complex, eluded, the 5' adapter was added, the Ded1-RNA complex was eluted, digested with protease K, the RNA was phenol/chloroform extracted, ethanol precipitated and then subjected to RT-PCR. The PCR products were combined and concentrated with a NucleoSpin Gel and PCR Clean-up kit amplification, purified on 6% PAGE and material migrating between ~120 and ~200 residues were recovered.