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RNA-Protein Immunoprecipitation Methods
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:
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Data from Lipchina I, Elkabetz Y, Hafner M, et al. Genome-wide identification of microRNA targets in human ES cells reveals a role for miR-302 in modulating BMP response. Genes Dev. 2011;25(20):2173-86.
This is a peak from an Ago2 PAR-CLIP experiment in human embryonic stem cells. The bars indicate the read count at each position, where red indicates a matching base and yellow indicates a mismatching base. As the scale bar indicates, libraries with extensive digestion ("footprinting"), traceable mutations, and known motifs allow the discovery of extremely narrow target sites at base pair resolution. Those additional criteria also help filter out false positives, which short RNA libraries are susceptible to since filtering for PCR duplicates is complicated by the expectation of low-complexity read distributions.