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

January 2021

Dimitrios G Anastasakis, Alexis Jacob, Parthena Konstantinidou, Kazuyuki Meguro, Duncan Claypool, Pavol Cekan, Astrid D Haase, Markus Hafner

The authors offer an improvement to the PAR-CLIP method that is more streamlined and does not require the use of radioactive labels. CLIP-seq (CrossLinking and ImmunoPrecipitation) is used to evaluate protein-RNA interactions during gene expression. PAR-CLIP uses in vivo labelling of RNA with photoreactive nucleosides . The method outlined in this protocol (fPAR-CLIP) uses direct ligation of fluorescent adapters to RNA/protein complexes, followed by the isolation of the RNA. The original PAR-CLIP method requires size fractionation on denaturing polyacrylamide gels. The fPAR-CLIP method presented here eliminates this requirement, is more sensitive, and is completed in 2 rather than 4 days.

Pippin Prep is used to size select PCR fragments and eliminate primers and adapters.

Author Affiliations:
Laboratory of Muscle Stem Cells and Gene Regulation, National Institute for Arthritis and Musculoskeletal and Skin Disease, NIH, Bethesda MD

Laboratory of Cellular and Molecular Biology, National Institutes of Diabetes and Digestive and Kidney Diseases, NIH, Bethesda MD

Laboratory of Clinical Immunology & Microbiology, National Institute of Allergy and Infectious Diseases, NIH, Bethesda MD

MultiplexDX, Bratislava, Slovakia

Nucleic Acids Research
DOI: 10.1093/nar/gkab011

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