title: Quantitative assessment of RNA-protein interactions with high-throughput sequencing-RNA affinity profiling.
abstract: Because RNA-protein interactions have a central role in a wide array of biological processes, methods that enable a quantitative assessment of these interactions in a high-throughput manner are in great demand. Recently, we developed the high-throughput sequencing-RNA affinity profiling (HiTS-RAP) assay that couples sequencing on an Illumina GAIIx genome analyzer with the quantitative assessment of protein-RNA interactions. This assay is able to analyze interactions between one or possibly several proteins with millions of different RNAs in a single experiment. We have successfully used HiTS-RAP to analyze interactions of the EGFP and negative elongation factor subunit E (NELF-E) proteins with their corresponding canonical and mutant RNA aptamers. Here we provide a detailed protocol for HiTS-RAP that can be completed in about a month (8 d hands-on time). This includes the preparation and testing of recombinant proteins and DNA templates, clustering DNA templates on a flowcell, HiTS and protein binding with a GAIIx instrument, and finally data analysis. We also highlight aspects of HiTS-RAP that can be further improved and points of comparison between HiTS-RAP and two other recently developed methods, quantitative analysis of RNA on a massively parallel array (RNA-MaP) and RNA Bind-n-Seq (RBNS), for quantitative analysis of RNA-protein interactions.
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Abdullah
authors.identifier: 0000000168612853
authors.initials: A
authors.lastname: Ozer
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: Jacob M
authors.initials: JM
authors.lastname: Tome
authors.affiliation: Molecular Microbial Pathogenesis Unit, Institut Pasteur, Paris, France.
authors.forename: Robin C
authors.initials: RC
authors.lastname: Friedman
authors.affiliation: Illumina, San Diego, California, USA.
authors.forename: Dan
authors.initials: D
authors.lastname: Gheba
authors.affiliation: Illumina, San Diego, California, USA.
authors.forename: Gary P
authors.initials: GP
authors.lastname: Schroth
authors.affiliation: Department of Molecular Biology and Genetics, Cornell University, Ithaca, New York, USA.
authors.forename: John T
authors.initials: JT
authors.lastname: Lis
mesh_terms: D001483:Base Sequence; D059014:High-Throughput Nucleotide Sequencing; D008969:Molecular Sequence Data; D011506:Proteins / Q000378:metabolism; D012313:RNA / Q000378:metabolism*
chemical_list: D011506:Proteins; D012313:RNA