Ribonucleoprotein (RNP) immunoprecipitation (IP), broadly, is an experimental protocol that isolates a protein and any associated RNA molecules from a mixed cellular lysate using an antibody with affinity for some portion of the target protein. Methods targeting RNA sequences in order to profile associated proteins by mass spectrometry exist, but are not nearly as common and not the subject of this class. Beyond these details, there exists wide variation between protocols used to reproducibly isolate RNP complexes and extract its RNA component for next-generation sequencing. While any number of protocol variants can give different experimental results, there are certain protocol parameters that are so significantly important to the end result that they can be thought of as protocol categories unto themselves. The following is a schematic of an example protocol representative of the most fundamental protocol, which we will call RIP-Seq:
Zhao J, Ohsumi TK, Kung JT, et al. Genome-wide identification of polycomb-associated RNAs by RIP-seq. Mol Cell. 2010;40(6):939-53.
Within this method, significant variation arises in several protocol steps, most commonly any fragmentation steps between RNA extraction and adaptor ligation, the size selected during gel purification, and the details of the Illumina sequencing protocol (single vs paired end, read length, etc). However, the most important detail is that, in the IP experiment, full-length RNA molecules are, in theory, recovered and no exogenous method is applied to covalently link RNA to protein - the "pull-down" uses only the strength of the endogenous interactions, and can be susceptible to associations that occur in vitro rather than in vivo.
A second category of protocols is distinguished by the use of a form of cross-linking, and can be broadly called CLIP-Seq (standing for Cross-Linking and ImmunoPrecipitation). Formaldehyde, a popular cross-linking reagent in other applications, is not used since the conditions necessary to reverse those cross-links (heating at 65°C for hours or overnight) rapidly degrades RNA. Instead, UV light is used, at varying frequencies. When UV light alone is used to cross-link, the protocol is often called HITS-CLIP (standing for HIgh Throughput Sequencing of CrossLinked and ImmunoPrecipitated RNA). In another protocol variant, a modified base (typically 4-thiouridine) is added to cells during growth, and is incorporated into RNA synthesis. This base, when exposed to UV light, forms cross-links with nearby protein molecules and mutates the RNA sequence at that position in a predictable manner. This is called PAR-CLIP (standing for PhotoActivatable Ribonucleoside CLIP). A third protocol variant, called iCLIP, involves circularization of the RNA molecule during library preparation, and can occur downstream of either HITS-CLIP or PAR-CLIP. The following diagram gives a sense of the relevant differences from a great review article:
König J, Zarnack K, Luscombe NM, Ule J. Protein-RNA interactions: new genomic technologies and perspectives. Nat Rev Genet. 2011;13(2):77-83.
This variation