ArticleJournal of visualized experiments : JoVE2019
Identification of Footprints of RNA:Protein Complexes via RNA Immunoprecipitation in Tandem Followed by Sequencing (RIPiT-Seq).
Article in Journal of visualized experiments : JoVE, 2019. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 3 papers.
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Who cites it
3 citing papers in PubMed, 3 citations in OpenAlex.
- RIPiT-Seq: A tandem immunoprecipitation approach to reveal global binding landscape of multisubunit ribonucleoproteins.Methods in enzymology · 2021Article
- Shaping the Innate Immune Response Through Post-Transcriptional Regulation of Gene Expression Mediated by RNA-Binding Proteins.Frontiers in immunology · 2021Review
- Chemical crosslinking enhances RNA immunoprecipitation for efficient identification of binding sites of proteins that photo-crosslink poorly with RNA.RNA (New York, N.Y.) · 2020Article
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Authors and funding
3 authors at 1 institution in 1 country.
Funding
Abstract
RNA immunoprecipitation in tandem (RIPiT) is a method for enriching RNA footprints of a pair of proteins within an RNA:protein (RNP) complex. RIPiT employs two purification steps. First, immunoprecipitation of a tagged RNP subunit is followed by mild RNase digestion and subsequent non-denaturing affinity elution. A second immunoprecipitation of another RNP subunit allows for enrichment of a defined complex. Following a denaturing elution of RNAs and proteins, the RNA footprints are converted into high-throughput DNA sequencing libraries. Unlike the more popular ultraviolet (UV) crosslinking followed by immunoprecipitation (CLIP) approach to enrich RBP binding sites, RIPiT is UV-crosslinking independent. Hence RIPiT can be applied to numerous proteins present in the RNA interactome and beyond that are essential to RNA regulation but do not directly contact the RNA or UV-crosslink poorly to RNA. The two purification steps in RIPiT provide an additional advantage of identifying binding sites where a protein of interest acts in partnership with another cofactor. The double purification strategy also serves to enhance signal by limiting background. Here, we provide a step-wise procedure to perform RIPiT and to generate high-throughput sequencing libraries from isolated RNA footprints. We also outline RIPiT's advantages and applications and discuss some of its limitations.
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Registered trials
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