Modified forms of easyCLIP

Abstract

The easyCLIP protocol describes a method for both normal CLIP library construction and the absolute quantification of RNA cross-linking rates, data which could be usefully combined to analyze RNA-protein interactions. Using these cross-linking metrics, significant interactions could be defined relative to a set of random non-RBPs. The original easyCLIP protocol did not use index reads, required custom sequencing primers, and did not have an easily reproducible analysis workflow. This short paper attempts to amend these deficiencies. It also includes some additional technical experiments and investigates the usage of alternative adapters. The results here are intended to allow more options to easily perform and analyze easyCLIP.

Analyzing RNA‐Protein Interactions by Cross‐Link Rates and CLIP‐seq Libraries

“…Distinguishing significant interactions from background is informed by the second method of easyCLIP, the determination of cross‐link efficiencies. Non‐RBPs often have cross‐link efficiencies in the 0.01%‐0.1% range, while RBPs are in the 0.1%‐10% range (Porter, Miao, et al., 2021). Combining the cross‐link efficiency and sequencing data allows for determining cross‐links per protein to specific RNAs or types of RNAs, which can then be compared with non‐RBPs to identify interactions more likely to be specific, with an example of this in Figure 1E (Porter, Miao, et al., 2021).…”

“…A cross‐link rate produced by Basic Protocol 2, using only RNA in the minimal region (Fig. 3B), where the protein of interest cross‐linked to a short oligo and ligated to the adapter is expected to run above 0.1% is indicative of an RBP (Porter, Miao, et al., 2021). However, a cross‐link rate alone is not a perfectly reliable indicator; sequencing libraries should also be prepared to determine if the RNA signal is high due to an artifact (Porter, Miao, et al., 2021).…”

“…In addition, this protocol is unnecessary if enough of the protein of interest runs in a crosslinked band that it can be directly quantified by western blot (the standard cutoff being 5 ng, see Janes, 2015). Fluorescence changes between experiments are mostly within two fold (Porter, Garg, et al, 2021) so once a standard is created it need not always be run.…”

“…Consider Figure 1E (adapted from Fig. 6d in Porter, Miao, et al., 2021), in which non‐RBPs could be misinterpreted to be small nucleolar RNA (snoRNA)‐binding proteins until cross‐link rates are included, at which point only the authentic snoRNA‐binding protein FBL is associated with snoRNA. The cross‐link rate adjustment avoids a similar error with tRNA (Fig.…”

“…Since the publication of easyCLIP, a few improvements have been released (Porter, Garg, et al., 2021). Specifically, (1) custom primer sequences were replaced with a hybrid of TruSeq and Nextera sequences to allow sequencing without custom primers; (2) PCR barcoding was added to decrease the number of expensive adapters needed; (3) the 3′ adapter was simplified and the need for pre‐adenylation removed to lower reagent cost; and (4) the need to conjugate a dye to the L5 adapter was replaced with the option of Cy5.5‐oligo synthesis.…”