Protocol

Nuclear Bodies and Noncoding RNAs

Volume 1262 of the series Methods in Molecular Biology pp 183-197

Date:

RNA Antisense Purification (RAP) for Mapping RNA Interactions with Chromatin

  • Jesse EngreitzAffiliated withBroad Institute of Harvard and MITDivision of Health Sciences and Technology, MIT Email author 
  • , Eric S. LanderAffiliated withDepartment of Biology, MITDepartment of Systems Biology, Harvard Medical School 
  • , Mitchell GuttmanAffiliated withDivision of Biology and Biological Engineering, California Institute of Technology 

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Abstract

RNA-centric biochemical purification is a general approach for studying the functions and mechanisms of noncoding RNAs. Here, we describe the experimental procedures for RNA antisense purification (RAP), a method for selective purification of endogenous RNA complexes from cell extracts that enables mapping of RNA interactions with chromatin. In RAP, the user cross-links cells to fix endogenous RNA complexes and purifies these complexes through hybrid capture with biotinylated antisense oligos. DNA loci that interact with the target RNA are identified using high-throughput DNA sequencing.

Key words

RNA purification Chromatin Localization lncRNA Hybrid capture