Single Oligonucleotide Capture of RNA And Temperature Elution Series ( SOCRATES ) for Identification of RNA-binding Proteins

Yu, Allen T, Aggarwal, Disha, Pappin, Darryl, Spector, David L (December 2022) Single Oligonucleotide Capture of RNA And Temperature Elution Series ( SOCRATES ) for Identification of RNA-binding Proteins. Bio-protocol, 12 (24). e4572. ISSN 2331-8325

Abstract

The importance of studying the mechanistic aspects of long non-coding RNAs is being increasingly emphasized as more and more regulatory RNAs are being discovered. Non-coding RNA sequences directly associate with generic RNA-binding proteins as well as specific proteins, which cooperate in the downstream functions of the RNA and can also be dysregulated in various physiologic states and/or diseases. While current methods exist for identifying RNA-protein interactions, these methods require high quantities of input cells or use pooled capture reagents that may increase non-specific binding. We have developed a method to efficiently capture specific RNAs using less than one million input cells. One single oligonucleotide is used to pull down the target RNA of choice and oligonucleotide selection is driven by sequence accessibility. We perform thermal elution to specifically elute the target RNA and its associated proteins, which are identified by mass spectrometry. Ultimately, two target and control oligonucleotides are used to create an enrichment map of interacting proteins of interest. This protocol was validated in: eLife (2021), DOI: 10.7554/eLife.68263.

Item Type:	Paper
Subjects:	bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > long non-coding RNA bioinformatics > genomics and proteomics > genetics & nucleic acid processing > DNA, RNA structure, function, modification > oligonucleotide bioinformatics > genomics and proteomics > genetics & nucleic acid processing > protein structure, function, modification > protein types > RNA binding protein
CSHL Authors:	Yu, Allen Aggarwal, Disha Pappin, Darryl J. Spector, David L.
Communities:	CSHL labs > Pappin lab CSHL labs > Spector lab
SWORD Depositor:	CSHL Elements
Depositing User:	CSHL Elements
Date:	20 December 2022
Date Deposited:	23 Jan 2023 21:02
Last Modified:	23 Jan 2023 21:02
PMCID:	PMC9797359
URI:	https://repository.cshl.edu/id/eprint/40801

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