High-throughput methodology for identifying RNA-protein interactions transcriptome-wide

摘要

Methods of identifying RNA-protein interaction sites are provided. Systems for identifying RNA-protein interaction sites are provided. Systems for identifying secondary structures are provided. Methods of identifying secondary structures are provided. Methods of identifying RNA-binding proteins are provided.

主权项

1. A method of transcriptome-wide identification of RNA-protein interaction sites, comprising:
(a) preparing an RNase footprinting library, wherein said preparation comprises:
i. exposing a eukaryotic cell to a crosslinking agent;ii. obtaining a nucleic acid-containing sample from the cell;iii. treating a first fraction of the sample with a single-stranded RNase;iv. thereafter treating the first fraction with a protease or proteinase;v. treating a second fraction of the sample with a double-stranded RNase;vi. thereafter treating the second fraction with a protease or proteinase;vii. isolating RNA from the first fraction and the second fraction; andviii. preparing a strand specific sequence library from the isolated RNA; (b) preparing an RNase control library, wherein said preparation comprises:
i. exposing a eukaryotic cell to a crosslinking agent;ii. obtaining a nucleic acid-containing sample from the cell;iii. treating a first fraction of the sample with protease or proteinase;iv. thereafter treating the first fraction of the sample with a single-stranded RNase;v. treating a second fraction of the sample with protease or proteinase;vi. thereafter treating the second fraction of the sample with a double-stranded RNase;vii. isolating RNA from the first fraction and the second fraction; andviii. preparing a strand specific sequence library from the isolated RNA, (c) identifying candidate protein binding sites within the RNase footprinting library; and (d) identifying sequence level motifs within the candidate protein binding sites.