Microbial iCLIP2: enhanced mapping of RNA–protein interaction by promoting protein and RNA stability

Optimized lysis buffer conditions. (A) Autoradiograph of Grp1-G-RNA (area of interest >45 kDa) complexes, purified with lysis buffer containing 2–8 M urea. (B) Autoradiograph of Rrm4/Rrm4^mR1-G-RNA complexes, immunoprecipitated with or without 2 M urea-containing lysis buffers. The area of interest is indicated (>112 kDa). The inclusion of 2 M urea in the lysis buffer results in a reduced overall radioactive signal, suggesting higher specificity. (C) The relative area quantification was determined by the signal intensity of the area of interest (purple) divided by the overall signal of the lane (marked black). Four independent biological replicates of Rrm4 and Rrm4^mR1-G/RNA-complexes, purified with and without 2 M urea-containing lysis buffer, were quantified (paired t-test; P-value: ** <0.01).