State-of-the-art methods to discover where RBPs bind rely on crosslinking and immunoprecipitation (CLIP) of the ribonucleoprotein complex, followed by sequencing. Such protocols are technically challenging, require radioactivity, have a high experimental failure rate, and generate a high percentage of duplicate reads. “When performing large-scale CLIP experiments as part of the ENCODE consortium, we determined that the current methods were not robust enough to profile hundreds of factors,” related Eric Van Nostrand and Gene Yeo from the University of California, San Diego by email. They and their colleagues created enhanced CLIP (eCLIP) to solve these problems.
eCLIP borrows its 2-step multiplex library preparation technique from ChIP-seq. First, the researchers ligate an indexed 3´ adapter (which allows for subsequent pooling) to the RNA fragments attached to immunoprecipitation beads. They then separate the complexes by electrophoresis and digest away the protein. After reverse transcribing the RNA, they ligate a second 3’ adapter containing an inline random 5- or 10-mer to the now single-stranded DNA before PCR amplification.
The fact that reverse transcription often terminates at the site of cross-linking allows the researchers to obtain nucleotide resolution. In the case of identical sequencing reads, the random single-stranded DNA adapter sequence will distinguish unique fragments from PCR duplicates, allowing the latter to be discarded, thus making the number of mapped reads more quantitative and minimizing wasted sequencing. In addition, eCLIP adds a size-matched pre-immunoprecipitation control for non-specific background and inherent biases.
These modifications mean that eCLIP requires about 1000-fold less amplification than its predecessors to achieve a higher percentage of useable reads and generate libraries with enough complexity to identify sequences used by more promiscuous RBPs. Now, it’s time to use these sequences to figure out how RBPs actually do their jobs.
Van Nostrand EL, Pratt GA, Shishkin AA, Gelboin-Burkhart C, Fang MY, Sundararaman B, Blue SM, Nguyen TB, Surka C, Elkins K, Stanton R, Rigo F, Guttman M, Yeo GW. Robust transcriptome-wide discovery of RNA-binding protein binding sites with enhanced CLIP (eCLIP). Nat Methods 2016 Mar 28.