RNA-Binding Protein Interactome Capture

Initial UV crosslinking and oligo(dT) purification followed the mRNA interactome capture protocol (Castello et al., 2013b (link)). Complete proteolytic digestions were performed with LysC or ArgC for 8 hr at 37°C. Polyadenylated RNA and crosslinked peptides were diluted in 20 mM Tris-HCl, 500 mM LiCl, 1 mM DTT, and 0.5 mM EDTA and recaptured on oligo(dT) beads. The supernatant was processed for MS (released peptides). oligo(dT) beads were washed as in Castello et al. (2013b (link)). All fractions were treated with trypsin and labeled with stable isotopes in vitro (Boersema et al., 2008 (link)). Peptides were analyzed on a liquid chromatography-tandem MS (LC-MS/MS) platform. The R-scripts used for the analyses can be found in the R/Bioconductor data-package RBDmapHeLa (http://www.bioconductor.org). RBDmap data can be accessed under http://www-huber.embl.de/users/befische/RBDmap.