RNA-binding proteins (RBPs) are central for gene expression by controlling the RNA fate from birth to decay. Various disorders arising from perturbations of RNA-protein interactions document their critical function. However, deciphering their function is complex, limiting the general functional elucidation of this growing class of proteins and their contribution to (patho)physiology.
Here, researchers from the University Medical Center Mainz present sCLIP, a simplified and robust platform for genome-wide interrogation of RNA-protein interactomes based on crosslinking-immunoprecipitation and high-throughput sequencing. sCLIP exploits linear amplification of the immunoprecipitated RNA improving the complexity of the sequencing-library despite significantly reducing the amount of input material and omitting several purification steps. Additionally, it permits a radiolabel-free visualization of immunoprecipitated RNA.
sCLIP—a simplified platform for studying RNA–protein interactomes by using crosslinking immunoprecipitation (CLIP) sequencing with a highly sensitive and non-radioactive biochemistry for low input material. (A) Schematic overview of the sCLIP technique.
Day 1: RNA–RBP interactions are preserved by in vivo UV-crosslinking. After crosslinking, the intact cells are lysed and the RNA not covered by crosslinked RBPs is partially digested. After immunoprecipitation an aliquot of the ribonucleoprotein (RNP) complexes is visualized by a non-radioactive labeling strategy (based on biotinylated ADP; see material and methods; Day 2, Following the IP the remaining material is digested with proteinase K and the bound RNA is released Next, the RNA is polyadenylated and then reversely transcribed by using a modified oligo d(T) primer that harbors an in line and a random barcode along with a sequencing platform-compatible Illumina adaptor and a T7 promotor (Day 3). Following reverse transcription (RT) the cDNA is in vitro transcribed and an Illumina adaptor is ligated to the mRNA 3΄end (Day 4). Finally, the amplified RNA is reversely transcribed and amplified with 10 cycles of PCR; afterward the libraries are subjected to high-throughput sequencing, and the sequencing data is analysed by an integrated sCLIP Data processing workflow.