PTex - Purification of Cross-linked RNA-Protein Complexes by Phenol-Toluol Extraction

PTex is a protocol to purify UV-cross-linked ribonucleoproteins (RNPs) in an unbiased and sequence-independent fashion. Total RNA from HEK293 cells was analysed by RNASeq from whole cell lysates as well as after purification of RNPs. Human HEK293 cells were in vivo UV-cross-linked with light at 254 nm wavelength and subjected to the PTex procedure (a modified phenol extraction) which will enrich for cross-linked RNA-protein hybrids and deplet non-cross-linked RNA and proteins. RNA from HEK293 cells was prepared (input total RNA control) as well as RNA purified in complexes (PTex samples). 3 different UV-cross-linking intensities were used (resulting in 4 conditions: no cross-link (noCL), 0.015 J/cm2 (0015), 0.15 J/cm2 (015) and 1.5 J/cm2 (15)).