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A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol

ABSTRACT: A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol

PROVIDER: PRJNA688225 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR13313325.fastq.gz	Fastqsanger.gz
	SRR13313326.fastq.gz	Fastqsanger.gz
	SRR13313327.fastq.gz	Fastqsanger.gz
	SRR13313328.fastq.gz	Fastqsanger.gz
	SRR13313329.fastq.gz	Fastqsanger.gz

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A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol

Project description:Streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. Protocol is based on direct ligation of a fluorescently labeled adapter to the 3’end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation time from 4-5 to 2 days and increases sensitivity by 10-100-fold.

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A non-radioactive, improved PAR-CLIP and small RNA cDNA library preparation protocol.

Project description:Crosslinking and immunoprecipitation (CLIP) methods are powerful techniques to interrogate direct protein-RNA interactions and dissect posttranscriptional gene regulatory networks. One widely used CLIP variant is photoactivatable ribonucleoside enhanced CLIP (PAR-CLIP) that involves in vivo labeling of nascent RNAs with the photoreactive nucleosides 4-thiouridine (4SU) or 6-thioguanosine (6SG), which can efficiently crosslink to interacting proteins using UVA and UVB light. Crosslinking of 4SU or 6SG to interacting amino acids changes their base-pairing properties and results in characteristic mutations in cDNA libraries prepared for high-throughput sequencing, which can be computationally exploited to remove abundant background from non-crosslinked sequences and help pinpoint RNA binding protein binding sites at nucleotide resolution on a transcriptome-wide scale. Here we present a streamlined protocol for fluorescence-based PAR-CLIP (fPAR-CLIP) that eliminates the need to use radioactivity. It is based on direct ligation of a fluorescently labeled adapter to the 3'end of crosslinked RNA on immobilized ribonucleoproteins, followed by isolation of the adapter-ligated RNA and efficient conversion into cDNA without the previously needed size fractionation on denaturing polyacrylamide gels. These improvements cut the experimentation by half to 2 days and increases sensitivity by 10-100-fold.

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