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Hfq RIL-seq analysis

ABSTRACT: Hfq RIL-seq analysis

PROVIDER: PRJNA816425 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR18331300_1.fastq.gz	Fastqsanger.gz
	SRR18331300_2.fastq.gz	Fastqsanger.gz
	SRR18331301_1.fastq.gz	Fastqsanger.gz
	SRR18331301_2.fastq.gz	Fastqsanger.gz
	SRR18331302_1.fastq.gz	Fastqsanger.gz

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Project description:A key challenge for understanding the role(s) played by short, non-coding RNAs (sRNAs) in bacteria is identifying the mRNA targets regulated by the sRNAs. Because the Hfq protein mediates the interactions between many sRNAs and the corresponding target mRNAs, one apporach to identify the mRNA targets of sRNAs is to capture sRNA:mRNA interactions occuring on Hfq by exposing cells to UV-irradiation, which forms cross-links between nucleic acids and proteins. We subjected cells of P. aeruginosa strain PAO1 and a derivative of PAO1 harboring a C-terminal VSV-G epitope on Hfq to UV-irradiation, immune-precipitated the Hfq-RNA complexes, ligated neighboring RNA molecules together with RNA Ligase, and then purififed the resulting RNAs. These RNAs were converted into cDNA libraries and sequenced using the Illumina NextSeq platform and then subjected to RIL-seq analysis pipeline (version 0.78) to identify chimeric RNA molecules. We also performed RNA-seq for PAO1 ∆phrS cells harboring an empty vector (pEV) or a vector expressing PhrS (pPhrS).

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Project description:Most E. coli sRNAs interact with their mRNA targets through simultaneous binding to the Hfq chaperon. In this experiment we cross-linked RNA to proteins in-vivo then did Hfq IP followed by ligation of bound RNAs and sequencing to identify sRNA-mRNA interactions. We termed the method RIL-seq for RNA Interactions by Ligation - sequencing.

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