easyCLIP Analysis of RNA-Protein Interactions Incorporating Absolute Quanti�cation

In general, an RNA-binding protein (RBP) may be considered a protein with an abnormally frequent interaction with RNA, and a "target RNA" for a speci�c protein may be considered an RNA with an abnormally frequent interaction with that protein. Traditional ultraviolet (UV) cross-linking immunoprecipitation (CLIP)-sequencing analysis methods generally de�ne interactions by methods that are indirectly assessing the latter. However, there has been limited direct assessment of these metrics by determining RNA cross-link rates or the RNA-binding pro�les of non-RBPs. Here, we describe a method to determine RNA cross-link rates and create sequencing libraries for a protein of interest, either of which may be performed on their own. easyCLIP is a relatively short, reliable, and e�cient CLIP method, with the capacity to directly visualize RNA libraries and diagnose methodological problems. By combining the sequencing data and cross-link rates, the absolute frequency of given cross-links may be compared between control and experimental proteins, to nominate potential RBPs and distinctive RNA-protein interactions.


Introduction
Currently, there is no general method to estimate absolute RNA-protein interaction frequencies and a quantitative test is needed to assess whether any non-random interaction with an RNA exists.The frequencies of RNA-protein complexes, per-cell and per-interaction partner, would enable the fundamental characterization of RNA-protein interaction networks.Determining the targets of an RBP by conventional approaches, such as enrichment over negative control immunopuri cation or by clustering of cross-links, are ultimately but indirectly determining if the absolute count of an RNA-protein complex in the cell is abnormally high.De ning RNA-protein interaction events per-cell and per-protein in absolute quantities, in contrast, may provide a framework for describing a global and widely reproducible view of RNA-protein interactions.
Here, we report a re nement of current CLIP protocols, termed easyCLIP.easyCLIP produces conventional CLIP-seq libraries, quanti es RNA cross-links-per-protein and provides visual con rmation of each step.29.Determine the amount of cross-linked RNA in immunopuri ed samples by multiplying uorescence by the fmols/ uorescence of the RNA standard.Let this amount be "R", calculated as R = (observed uorescence for RNA) * F.

Reagents
30.Add the fmols of cross-linked RNA to the fmols of monomeric, un-cross-linked protein to obtain the total amount of protein.Let this amount be "Pt", calculated as Pt = R + P.
31.Divide the fmols of RNA by the total amount of protein to obtain the fraction cross-linked and ligated.This is calculated as R/Pt.
Combination cross-link rate with sequencing date For a given location or RNA, the number of reads in a CLIP library at that location or RNA as a fraction of all reads is the fraction of cross-links at that RNA or location.This fraction may be multiplied by the cross-links per protein to determine the cross-links per protein at the given location or RNA.

Troubleshooting
RNAse: by uorescence on nitrocellulose (NC): under-digested sample runs at the top of the gel, and overdigested sample has no smear upward in the minimal region.It is easy to over-digest RNA so that it has a very low mapping rate, but relatively di cult to over-digest so much that the 5' ligation does not occurthis could be caused by the RNAse and ligase having similar requirements for substrate length.RNAse levels may work better for library constructions for many proteins at ~3 minutes digestion at 0.01 U/ul RNAse.

5' and 3' ligations:
Assessed by protein shifts and total uorescence on NC.If both adapters are labeled, the success of the ligations is directly visualized by the amount of uorescence.Single vs dual protein shifting is visible with some proteins, and is the most sensitive (and by far the easiest) of the three methods we have outlined for ligation e ciency estimates.