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Series GSE95851 Query DataSets for GSE95851
Status Public on Mar 10, 2017
Title Comprehensive and quantitative mapping of RNA-protein interactions across a transcribed eukaryotic genome
Organism Saccharomyces cerevisiae
Experiment type Other
Summary Purpose: High-throughput sequencing has transformed modern biology, but its repertoire is currently confined to reading DNA molecules. Here, we report hardware and software adaptations that allow the very methods that enabled the genomic sequencing revolution to be applied to fluorescence-based biochemical assays, on a massive scale.
Methods: Using commonly available hardware, we built a customizable, open-source platform that leverages the inherent throughput of Illumina technology for direct biophysical measurements. We used the platform to quantitatively measure the binding affinity of the prototypical RBP Vts1 for every transcript in the S. cerevisiae genome.
Results: Our transcribed genome array (TGA) assayed both rare and abundant transcripts with equivalent proficiency, revealing hundreds of low-abundance targets missed by previous approaches. These targets regulated diverse biological processes including nutrient sensing and the DNA damage response, and implicated Vts1 in the ‘birth’ of new genes. TGA provided single nucleotide resolution for each binding site and delineated a highly specific sequence and structure motif for Vts1 binding.
Conclusions: Our technology establishes a flexible new platform for high-throughput biochemistry that can be easily extended to any nucleic acid template (e.g. the human exome), used to study diverse types of biochemical interaction (e.g. RNA-guided nucleases), and adapted to even higher- throughput systems (e.g. HiSeq). Our application of TGA to Vts1 i) doubled the number of known Vts1 targets, identifying key regulators of cell cycle and the DNA damage response ii) provided a marked improvement in the specificity of the protein’s binding motif, iii) generated structural insight into its ability to discriminate among targets, and iv) suggested that Vts1 may have a role in regulating the transcripts of evolutionarily nascent genes. The breadth of new findings stemming from analysis of an already exceptionally well-studied RBP suggests that TGA technology will be similarly enabling for other RBPs and establishes a new paradigm for quantitative, ultra high-throughput biochemistry.
 
Overall design A yeast genome sequenced on a MiSeq flow cell with an RNA polymerase promoter and stall sequence attached as adaptors.
 
Contributor(s) She R, Jarosz D
Citation(s) 28325876
Submission date Mar 09, 2017
Last update date May 15, 2019
Contact name Richard She
Organization name Whitehead Institute
Lab Weissman Lab
Street address 455 Main St Rm 639, Whitehead Institute
City Cambridge
State/province Massachusetts
ZIP/Postal code 02142
Country USA
 
Platforms (1)
GPL17143 Illumina MiSeq (Saccharomyces cerevisiae)
Samples (1)
GSM2527046 Log-phase yeast cells
Relations
BioProject PRJNA378598
SRA SRP101616

Download family Format
SOFT formatted family file(s) SOFTHelp
MINiML formatted family file(s) MINiMLHelp
Series Matrix File(s) TXTHelp

Supplementary file Size Download File type/resource
GSE95851_128nM_SAM_mask.txt.gz 209.3 Kb (ftp)(http) TXT
GSE95851_RAW.tar 14.4 Mb (http)(custom) TAR (of FASTQ)
GSE95851_SupplementaryTable3.xlsx 115.0 Kb (ftp)(http) XLSX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file
Processed data are available on Series record

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