We recently developed a protocol for the transcriptome-wide isolation of RNA recognition elements readily applicable to any protein or ribonucleoprotein complex directly contacting RNA (including RNA helicases, polymerases, or nucleases) expressed in cell culture models either naturally or ectopically (Hafner et al., 2010). Briefly, immunoprecipitation of the RNA-binding protein of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. In the course of lysate preparation and immunoprecipitation, the mRNAs are partially degraded using Ribonuclease T1. The isolated crosslinked RNA fragments are converted into a cDNA library and deep-sequenced using Solexa technology (see Explanatory Chapter: Next Generation Sequencing). By introducing photoreactive nucleosides that generate characteristic sequence changes upon crosslinking (see below), our protocol allows one to separate RNA segments bound by the protein of interest from the background un-crosslinked RNAs.
Keywords: 3′- and 5′-adapter ligation; Cell lysate preparation; Crosslinking and immunoprecipitation (CLIP) protocol; Immunoprecipitation and second RNase T1 digestion; PCR amplification of cDNA library; Posttranscriptional regulation (PTR); Protein G magnetic beads; Proteinase K digestion; UV crosslinking of 4-thiouridine-labeled cells; cDNA library preparation; cDNA library preparation/reverse transcription.
© 2014 Elsevier Inc. All rights reserved.